DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 285
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 285
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
TUMOR TARGETING CONJUGATES AND METHODS OF USE THEREOF
PRIORITY
[0001] This application claims the benefit of priority under 35 U.S.C.
119(e) to U.S.
Provisional Application Nos. 62/451,624, filed January 27, 2017; 62/481,867,
filed April 5,
2017; and 62/573,626, filed October 17, 2017, each of which are incorporated
herein by
reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been
submitted
electronically in ASCII format and is hereby incorporated by reference in its
entirety. Said
ASCII copy, created on January 26, 2018, is named 50358-714 601 SL.txt and is
4,346,727
bytes in size.
BACKGROUND
[0003] One of the leading causes of death in the United States is cancer. The
conventional
methods of cancer treatment, like chemotherapy, surgery, or radiation therapy,
tend to be either
highly toxic or nonspecific to a cancer, or both, resulting in limited
efficacy and harmful side
effects. However, the immune system has the potential to be a powerful,
specific tool in fighting
cancers. In many cases tumors can specifically express genes whose products
are required for
inducing or maintaining the malignant state. These proteins may serve as
antigen markers for the
development and establishment of more specific anti-cancer immune response.
The immune
response may include the recruitment of immune cells that target tumors
expressing these antigen
markers. Additionally, the immune cells may express genes whose products are
important to
proper immune function and may serve as markers for specific types of immune
cells. The
boosting of this specific immune response has the potential to be a powerful
anti-cancer
treatment that can be more effective than conventional methods of cancer
treatment and can have
fewer side effects.
INCORPORATION BY REFERENCE
[0004] All publications, patents, and patent applications mentioned in this
specification are
herein incorporated by reference to the same extent as if each individual
publication, patent, or
patent application was specifically and individually indicated to be
incorporated by reference.
1
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
SUMMARY
[0005] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; b) an effector antigen binding domain, wherein the
effector antigen binding
domain specifically binds to an antigen on an antigen presenting cell and
wherein the antigen is a
molecule on the antigen presenting cell; c) an Fc comprising domain; and d) an
immune-
stimulatory compound attached to the recombinant bispecific antibody by a
linker; wherein the
recombinant bispecific antibody induces greater immune cell activation when
the recombinant
bispecific antibody is bound to the tumor associated antigen and to the
antigen on the antigen
presenting cell as compared to when the recombinant bispecific antibody is
bound to the antigen
on the antigen presenting cell but not to the tumor associated antigen.
[0006] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; b) an effector antigen binding domain, wherein the
effector antigen binding
domain specifically binds to an antigen on an antigen presenting cell and is
an antibody antigen
binding domain, wherein the antigen is a molecule on the antigen presenting
cell; and c) a
domain comprising an Fc region; wherein the recombinant bispecific antibody
induces greater
immune cell activation when the recombinant bispecific antibody is bound to
the tumor
associated antigen and to the antigen on the antigen presenting cell as
compared to when the
recombinant bispecific antibody is bound to the antigen on the antigen
presenting cell but not to
the tumor associated antigen.
[0007] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; b) an effector antigen binding domain, wherein the
effector antigen binding
domain specifically binds to an antigen on an antigen presenting cell and is
an antibody antigen
binding domain, wherein the antigen is a molecule on the antigen presenting
cell; and c) a
domain comprising an Fc region; wherein the recombinant bispecific antibody
induces greater
immune cell activation in the presence of cells having cell surface tumor
associated antigen and
antigen presenting cells having cell surface antigen as compared to immune
cell activation in the
absence of cells having cell surface tumor associated antigen.
[0008] In some aspects, a recombinant bispecific antibody, comprising: a) a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; b) an effector antigen binding domain, wherein the
effector antigen binding
domain specifically binds to an antigen on an antigen presenting cell and
wherein the antigen is a
2
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
molecule on the antigen presenting cell; and c) an Fc comprising domain; and
d) an immune-
stimulatory compound attached to the recombinant bispecific antibody by a
linker; wherein the
recombinant bispecific antibody induces greater immune cell activation in the
presence of cells
having cell surface tumor associated antigen and antigen presenting cells
having cell surface
antigen as compared to immune cell activation in the absence of cells having
cell surface tumor
associated antigen. In some embodiments, the immune cell activation is
measured by a cytokine
release assay. In some embodiments, the immune cell activation by the
recombinant bispecific
antibody when the recombinant bispecific antibody is bound to the tumor
associated antigen and
to the antigen on the antigen presenting cell is at least two times, five
times, or ten times greater
than immune activation by the recombinant bispecific antibody when the
recombinant bispecific
antibody is bound to the antigen on the antigen presenting cell but not to the
tumor associated
antigen as measured by the cytokine release assay. In some embodiments, the
immune cell
activation by the recombinant bispecific antibody in the presence of cells
having cell surface
tumor associated antigen and antigen presenting cells having cell surface
antigen is at least two
times, five times, or ten times greater than immune cell activation by the
recombinant bispecific
antibody in the absence of the cells having cell surface tumor associated
antigen as measured by
the cytokine release assay. In some embodiments, the immune cell activation
comprises an
increase in one or more of: a) a secretion of one or more cytokines as
measured by the cytokine
release assay, b) a secretion of one or more chemokines as measured by an
ELISA immunoassay,
c) an expression level of one or more cell surface proteins associated with
immune stimulation as
measured by FACS, and d) an activity of one or more immune cell functions. In
some
embodiments, the activity of one or more immune cell functions comprises
antibody-dependent
cell-mediated cytotoxicity as measured by an ADCC assay, antibody dependent
cellular
phagocytosis as measured by an ADCP assay, or antigen cross-presentation as
measured by a
cross-presentation assay. In some embodiments, the recombinant bispecific
antibody induces
tumor-cell directed antibody-dependent cell-mediated cytotoxicity. In some
embodiments, the Fc
comprising domain has one or more amino acid substitutions that decrease the
binding affinity to
one or more Fey receptors as compared to a wild-type Fc comprising domain. In
some
embodiments, the effector antigen binding domain has an increased binding
affinity to the
antigen on the antigen presenting cell as compared to the binding affinity of
the effector antigen
binding domain of an antibody that lacks the target antigen binding domain. In
some
embodiments, a Kd of the binding affinity of the effector antigen binding
domain of the
recombinant bispecific antibody to the antigen on the antigen presenting cell
is increased by two
times, five times, ten times, fifty times, or one-hundred times compared to
the binding affinity of
3
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
the effector antigen binding domain of an antibody that lacks the target
antigen binding domain.
In some embodiments, a Kd for binding of the effector antigen binding domain
to the antigen on
the antigen presenting cell is less than 20 nM, less than 100 nM, or less than
500 nM. In some
embodiments, the Fc comprising domain is linked to the target antigen binding
domain and to the
effector antigen binding domain. In some embodiments, the target antigen
binding domain
comprises an immunoglobulin heavy chain variable region or antigen binding
fragment thereof
and an immunoglobulin light chain variable region or antigen binding fragment
thereof. In some
embodiments, the target antigen binding domain comprises a single chain
variable region
fragment (scFv). In some embodiments, the tumor associated antigen is an
antigen selected from
the group consisting of CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-
1,
HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-
binding
protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2,
GD3, GM2, Leg,
CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, a
metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1,
LMP2, HPV E6,
HPV E7, EGFRvIII, Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, MelanA/MART1,
Ras
mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a
Sarcoma
translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17,
PAX3, ALK,
androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1,
mesothelin
(MSLN), PSCA, MAGE Al, MAGE-A3, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn,
GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-
TESL Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie
3,
Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EPCAM, CA6,
NAPI2B, TROP2, CLDN18.2, fibroblast activation protein (FAP), RON, LY6E, FRA,
DLL3,
PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PD-L1, CD204, CD206,
CD301,
VTCN1, and VISTA. In some embodiments, the tumor associated antigen is
Her2/neu or
p185HER2. In some embodiments, the target antigen binding domain comprises the
following
CDRs: a) HCDR1 comprising an amino acid sequence of SEQ ID NO: 13; b) HCDR2
comprising an amino acid sequence of SEQ ID NO: 14; c) HCDR3 comprising an
amino acid
sequence of SEQ ID NO: 15; d) LCDR1 comprising an amino acid sequence of SEQ
ID NO: 18;
e) LCDR2 comprising an amino acid sequence of SEQ ID NO: 19; and f) LCDR3
comprising an
amino acid sequence of SEQ ID NO: 20; and wherein the recombinant bispecific
antibody
specifically binds to Her2/neu or p185HER2. In some embodiments, the target
antigen binding
domain comprises: a) a VH sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 12; and b) a VL sequence having at least 80% sequence
identity to an
4
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
amino acid sequence of SEQ ID NO: 17. In some embodiments, the target antigen
binding
domain comprises: a) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 11; and b) a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 16. In some embodiments, the
target antigen
binding domain comprises at least 80% sequence identity to the amino acid
sequence between
amino acid 20 and amino acid 110 of SEQ ID NO: 12 and at least 80% sequence
identity to the
amino acid sequence between amino acid 20 and amino acid 105 of SEQ ID NO: 17;
and
wherein the recombinant bispecific antibody specifically binds to Her2/neu or
p185HER2. In
some embodiments, the effector antigen binding domain comprises an
immunoglobulin heavy
chain variable region or antigen binding fragment thereof and an
immunoglobulin light chain
variable region or antigen binding fragment thereof. In some embodiments, the
effector antigen
binding domain comprises a single chain variable region fragment (scFv). In
some embodiments,
the scFv comprises at least 80% sequence identity to an amino acid sequence of
SEQ ID NO:
1312. In some embodiments, the antigen presenting cell is a dendritic cell. In
some embodiments,
the antigen on the antigen presenting cell is a costimulatory molecule. In
some embodiments, the
antigen on the antigen presenting cell is selected from the group consisting
of CD40, OX4OL,
DEC-205, 4-1BBL, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2,
CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, or BDCA-2. In some
embodiments, the effector antigen binding domain is a CD40 agonist. In some
embodiments, the
effector antigen binding domain comprises the following CDRs: a) HCDR1
comprising an amino
acid sequence of SEQ ID NO: 3; b) HCDR2 comprising an amino acid sequence of
SEQ ID NO:
4; c) HCDR3 comprising an amino acid sequence of SEQ ID NO: 5; d) LCDR1
comprising an
amino acid sequence of SEQ ID NO: 8; e) LCDR2 comprising an amino acid
sequence of SEQ
ID NO: 9; and f) LCDR3 comprising an amino acid sequence of SEQ ID NO: 10. In
some
embodiments, the effector antigen binding domain comprises: a) a VH sequence
having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 2; and b) a VL
sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
7. In some
embodiments, the effector antigen binding domain comprises: a) a heavy chain
sequence having
at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1; and
b) a light chain
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
6. In some
embodiments, the antigen on the antigen presenting cell is TREM2 or TNFR2. In
some
embodiments, the Fc comprising domain is linked C-terminal to the target
antigen binding
domain and N-terminal to the effector antigen binding domain. In some
embodiments, the Fc
comprising domain comprises one or more amino acid substitutions that reduce
the affinity of the
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Fc comprising domain to an Fc receptor compared to the affinity of a reference
Fc comprising
domain to the Fc receptor in the absence of the one or more amino acid
substitutions. In some
embodiments, reference Fc comprising domain is selected from the group
consisting of an Fc
comprising domain having the amino acid sequence of SEQ ID NO: 1314, SEQ ID
NO: 1315,
SEQ ID NO: 1316, and SEQ ID NO: 1317. In some embodiments, reference Fc
comprising
domain comprises an amino acid sequence selected from the group consisting of
SEQ ID NO:
1319, SEQ ID NO: 1320, SEQ ID NO: 1321, and SEQ ID NO: 1322. In some
embodiments, the
Fc comprising domain comprises a human IgGi Fc Region. In some embodiments,
the one or
more amino acid substitutions comprise L234A, L235A, G237A, and K322A,
according to the
EU index of Kabat. In some embodiments, the one or more amino acid
substitutions comprise
E233P, L234V, L235A, AG236, A327G, A3305, and P33 1S, according to the EU
index of
Kabat. In some embodiments, the Fc comprising domain comprises a human IgG2 Fc
Region. In
some embodiments, the one or more amino acid substitutions comprises K322A,
according to the
EU index of Kabat. In some embodiments, the Fc comprising domain comprises a
human IgG2a
Fc Region. In some embodiments, the one or more amino acid substitutions
comprises L235E,
E318A, K320A, K322A, according to the EU index of Kabat. In some embodiments,
the Fc
comprising domain is an Fc null. In some embodiments, the Fc comprising domain
has the amino
acid sequence of SEQ ID NO: 1313. In some embodiments, the Fc comprising
domain comprises
the amino acid sequence of SEQ ID NO: 1318. In some embodiments, the Fc
comprising domain
is linked C-terminal to the target antigen binding domain and has the amino
acid sequence of
SEQ ID NO: 1311. In some embodiments, the linker links the immune-stimulatory
compound to
the Fc comprising domain. In some embodiments, the recombinant bispecific
antibody further
comprises an immune stimulatory compound and a linker, wherein the linker
links the immune-
stimulatory compound to the Fc comprising domain. In some embodiments, the
immune-
stimulatory compound is a damage-associated molecular pattern molecule or a
pathogen-
associated molecular pattern molecule. In some embodiments, the immune-
stimulatory
compound is a Toll-like receptor agonist, STING agonist, or RIG-I agonist. In
some
embodiments, the immune-stimulatory compound is a CpG oligonucleotide, Poly
G10, Poly G3,
Poly I:C, Lipopolysaccharide, zymosan, flagellin, Pam3CSK4, PamCysPamSK4,
dsRNA, a
diacylated lipopeptide, a triacylated lipoprotein, lipoteichoic acid, a
peptidoglycan, a cyclic
dinucleotide, a 5'ppp-dsRNA, S-27609, CL307, UC-IV150, imiquimod, gardiquimod,
resiquimod, motolimod, VTS-1463G5-9620, G5K2245035, TMX-101, TMX-201, TMX-202,
isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240,
KU34B,
5B9200, SB11285, 8-substituted imidazo[1,5-a]pyridine, or CL663. In some
embodiments, the
6
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
immune-stimulatory compound is an inhibitor of TGFB, Beta-Catenin, PI3K-beta,
STAT3, IL-
10, IDO, or TDO. In some embodiments, the immune-stimulatory compound is
LY2109761,
GSK263771, iCRT3, iCRT5, iCRT14, LY2090314, CGX-1321, PRI-724, BC21,
ZINCO2092166, LGK974, IWP2, LY3022859, LY364947, SB431542, AZD8186, SD-208,
indoximod (NLG8189), F001287, GDC-0919, epacadostat (INCB024360), RG70099, 1-
methyl-
L-tryptophan, methylthiohydantoin tryptophan, brassinin, annulin B, exiguamine
A, PIM, LM10,
8-substituted 2-amino-3H-benzo[b]azepine-4-carboxamide, or INCB023843. In some
embodiments, the immune-stimulatory compound does not reduce the affinity of
the recombinant
bispecific antibody for binding to the tumor associated antigen or to the
antigen on the antigen
presenting cell. In some embodiments, the recombinant bispecific antibody
further comprises a
chemotherapeutic compound and a linker, wherein the linker links the
chemotherapeutic
compound to the Fc comprising domain. In some embodiments, the
chemotherapeutic compound
comprises an alkylating agent, an anthracycline, a cytoskeletal disruptor, a
histone deacetylase
inhibitor, an inhibitor of, a kinase inhibitor, a nucleoside analog or
precursor analog, a peptide
antibiotic, a platinum-based compound, or a plant alkaloid.
[0009] In some aspects, method of making a recombinant bispecific antibody
comprises: a)
producing an antibody construct comprising: i) a target antigen binding
domain, wherein the
target antigen binding domain specifically binds to a tumor associated
antigen; ii) an effector
antigen binding domain, wherein the effector antigen binding domain
specifically binds to an
antigen on an antigen presenting cell and the antigen is a molecule on the
antigen presenting cell,
wherein the antigen is a molecule on the antigen presenting cell; iii) an Fc
comprising domain;
and b) linking an immune-stimulatory compound to the antibody construct,
wherein the
recombinant bispecific antibody induces greater immune cell activation when
the recombinant
bispecific antibody is bound to the tumor associated antigen and to the
antigen on the antigen
presenting cell as compared to when the recombinant bispecific antibody is
bound to the antigen
on the antigen presenting cell but not to the tumor associated antigen.
[0010] In some aspects, a pharmaceutical composition comprises any recombinant
bispecific
antibody as described herein and a pharmaceutically acceptable carrier.
[0011] In some aspects, method of treating a subject in need thereof,
comprising administering
to the subject a therapeutic dose of any recombinant bispecific antibody as
described herein or
the pharmaceutical composition of any recombinant bispecific antibody as
described herein. In
some embodiments, the subject has cancer. In some embodiments, the recombinant
bispecific
antibody or the pharmaceutical composition is administered intravenously,
cutaneously,
subcutaneously, or injected at a site of affliction. In some embodiments, the
recombinant
7
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
bispecific antibody induces greater immune activation against a cancer as
measured by a
decrease in cancer cell number or volume as compared to non-cancerous tissue.
In some
embodiments, the recombinant bispecific antibody is administered intravenously
to the subject at
a minimum anticipated biological effect level of the recombinant bispecific
antibody, a biological
effect of the recombinant bispecific antibody is greater when the recombinant
bispecific antibody
is bound to the tumor associated antigen and to the antigen on the antigen
presenting cell as
compared to the biological effect of the recombinant bispecific antibody when
it is not bound to
the tumor associated antigen but is bound to the antigen on the antigen
presenting cell; and
wherein the biological effect is immune activation as measured by one or more
of the group
selected from secretion of one or more cytokines, secretion of one or more
chemokines,
expression level of one or more cell surface proteins associated with immune
stimulation,
antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular
phagocytosis, and
antigen cross-presentation. In some embodiments, the recombinant bispecific
antibody is
administered intravenously to the subject at the minimum anticipated
biological effect level of
the recombinant bispecific antibody, it induces a greater biological effect at
the site of the cancer
than at a non-cancerous site and wherein the biological effect is immune
activation as measured
by one or more of the group selected from secretion of one or more cytokines,
secretion of one or
more chemokines, expression level of one or more cell surface proteins
associated with immune
stimulation, antibody-dependent cell-mediated cytotoxicity, antibody dependent
cellular
phagocytosis, and antigen cross-presentation.
[0012] In some aspects, a conjugate comprises: a) an antibody construct
comprising: i) first
binding domain, wherein the first binding domain specifically binds to a tumor
antigen; ii) a
second binding domain, wherein the second binding domain specifically binds to
an antigen on
an antigen presenting cell, wherein the antigen is a molecule on the antigen
presenting cell; and
iii) an Fc domain; b) an immune-stimulatory compound; and c) a linker
attaching the antibody
construct to the immune-stimulatory compound, wherein the linker is covalently
bound to the
antibody construct and the linker is covalently bound to the immune-
stimulatory compound, and
wherein a molar ratio of immune-stimulatory compound to antibody construct is
less than 8;
wherein the first binding domain is attached to the Fc domain and the second
binding domain is
attached to the Fc domain or to a C-terminal end of a light chain of the first
binding domain;
wherein a Kd for binding of the Fc domain to an Fc receptor in a presence of
the first binding
domain and the second binding domain is no greater than about 100 times a Kd
for binding of the
Fc domain to the Fc receptor in an absence of the second binding domain; and
wherein immune
cell activation caused by the conjugate when bound to the tumor antigen as
measured by a
8
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cytokine release assay is greater than immune cell activation in the absence
of binding to the
tumor antigen.
[0013] In some aspects, a conjugate comprises: a) an antibody construct
comprising: i) first
binding domain, wherein the first binding domain specifically binds to a tumor
antigen; ii) a
second binding domain, wherein the second binding domain specifically binds to
an antigen on
an antigen presenting cell, wherein the antigen is a molecule on the antigen
presenting cell; and
iii) an Fc domain; b) an immune-stimulatory compound; and c) a linker
attaching the antibody
construct to the immune-stimulatory compound, wherein the linker is covalently
bound to the
antibody construct and the linker is covalently bound to the immune-
stimulatory compound, and
wherein a molar ratio of immune-stimulatory compound to antibody construct is
less than 8;
wherein the first binding domain is attached to the Fc domain and the second
binding domain is
attached to the Fc domain or to a C-terminal end of a light chain of the first
binding domain;
wherein a Kd for binding of the Fc domain to an Fc receptor in a presence of
the first binding
domain and the second binding domain is no greater than about 100 times a Kd
for binding of the
Fc domain to the Fc receptor in an absence of the second binding domain;
andwherein antigen
presenting cells are conditionally activated when the conjugate is bound to
the tumor antigen as
measured by a cytokine release assay.
[0014] In some aspects, an antibody construct comprises: a) a first binding
domain, wherein the
first binding domain specifically binds to a tumor antigen; b) a second
binding domain, wherein
the second binding domain specifically binds to an antigen on an antigen
presenting cell, wherein
the antigen is a molecule on the antigen presenting cell; and c) an Fc domain;
wherein the first
binding domain is attached to the Fc domain and the second binding domain is
attached to the Fc
domain or to a C-terminal end of a light chain of the first binding domain,
and wherein a Kd for
binding of the Fc domain to an Fc receptor in a presence of the first binding
domain and the
second binding domain is no greater than about 100 times a Kd for binding of
the Fc domain to
the Fc receptor in an absence of the second binding domain.
[0015] In some aspects, an antibody construct for use in inducing immune cell
activation
comprising: a) a first binding domain, wherein the first binding domain
specifically binds to a
tumor antigen; b) a second binding domain, wherein the second binding domain
specifically
binds to an antigen on an antigen presenting cell, wherein the antigen is a
molecule on the
antigen presenting cell; and c) an Fc domain; wherein the first binding domain
is attached to the
Fc domain and the second binding domain is attached to the Fc domain or to a C-
terminal end of
a light chain of the first binding domain, and wherein a Kd for binding of the
Fc domain to an Fc
receptor in a presence of the first binding domain and the second binding
domain is no greater
9
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
than about 100 times a Kd for binding of the Fc domain to the Fc receptor in
an absence of the
second binding domain; and wherein immune cell activation caused by the
antibody construct
upon binding to tumor antigen as measured by a cytokine release assay is
greater than immune
cell activation caused by the antibody construct in the absence of binding to
tumor antigen.
[0016] In some aspects, a conjugate for use in inducing immune cell activation
comprising: a)
an antibody construct comprising: i) first binding domain, wherein the first
binding domain
specifically binds to a tumor antigen; ii) a second binding domain, wherein
the second binding
domain specifically binds to an antigen on an antigen presenting cell, wherein
the antigen is a
molecule on the antigen presenting cell; and iii) an Fc domain; b) an immune-
stimulatory
compound; and c) a linker attaching the antibody construct to the immune-
stimulatory
compound, wherein the linker is covalently bound to the antibody construct and
the linker is
covalently bound to the immune-stimulatory compound, and wherein a molar ratio
of immune-
stimulatory compound to antibody construct is less than 8; wherein the first
binding domain is
attached to the Fc domain and the second binding domain is attached to the Fc
domain or to a C-
terminal end of a light chain of the first binding domain; wherein a Kd for
binding of the Fc
domain to an Fc receptor in a presence of the first binding domain and the
second binding
domain is no greater than about 100 times a Kd for binding of the Fc domain to
the Fc receptor in
an absence of the second binding domain; and wherein immune cell activation
caused by the
conjugate when bound to the tumor antigen as measured by a cytokine release
assay is greater
than immune cell activation in the absence of binding to the tumor antigen.
[0017] In some aspects, a conjugate for use in conditionally activating an
antigen presenting cell
comprising: a) an antibody construct comprising: i) first binding domain,
wherein the first
binding domain specifically binds to a tumor antigen; ii) a second binding
domain, wherein the
second binding domain specifically binds to an antigen on the antigen
presenting cell, and iii) an
Fc domain; b) an immune-stimulatory compound; and c) a linker attaching the
antibody construct
to the immune-stimulatory compound, wherein the linker is covalently bound to
the antibody
construct and the linker is covalently bound to the immune-stimulatory
compound, and wherein a
molar ratio of immune-stimulatory compound to antibody construct is less than
8; wherein the
first binding domain is attached to the Fc domain and the second binding
domain is attached to
the Fc domain or to a C-terminal end of a light chain of the first binding
domain; wherein a Kd
for binding of the Fc domain to an Fc receptor in a presence of the first
binding domain and the
second binding domain is no greater than about 100 times a Kd for binding of
the Fc domain to
the Fc receptor in an absence of the second binding domain; and wherein
antigen presenting cells
are conditionally activated when the conjugate is bound to the tumor antigen
as measured
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cytokine release assay. In some embodiments, a Kd for binding of the first
binding domain to the
tumor antigen in the presence of the immune-stimulatory compound is no greater
than about two
times, five times, ten times, or fifty times a Kd for binding of the first
binding domain to the
tumor antigen in an absence of the immune-stimulatory compound In some
embodiments, a Kd
for binding of the second binding domain to the antigen on the antigen
presenting cell in the
presence of the immune-stimulatory compound is no greater than about two
times, five times, ten
times, or fifty times a Kd for binding of the second binding domain to the
antigen on the antigen
presenting cell in an absence of the immune-stimulatory compound. In some
embodiments, a Kd
for binding of the first binding domain to the tumor antigen is no greater
than about 100 nM. In
some embodiments, a Kd for binding of the second binding domain to the antigen
on an antigen
presenting cell is no greater than about 100 nM. In some embodiments, an amino
acid sequence
of the tumor antigen has at least 80% sequence identity with the amino acid
sequence of a tumor
antigen selected from the group consisting of HER2, IL-2 receptor, EGFRvIII
(de2-7 EGFR),
EGFR, fibroblast activation protein (FAP), tenascin, a metalloproteinase,
endosialin, vascular
endothelial growth factor, avf33, WT1, LMP2, HPV E6, HPV E7, Her-2/neu, p53
nonmutant,
NY-ES0-1, GLP-3, MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl,
tyrosinase,
survivin, PSA, hTERT, a Sarcoma translocation breakpoint fusion protein,
EphA2, PAP, ML-
IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-
2,
mesothelin (MSLN), PSCA, MAGE Al, MAGE-A3, CYP1B1, PLAV1, BORIS, ETV6-AML,
NY-BR-1, RGS5, SART3, Carbonic anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17,
LCK,
MAGE C2, MAGE A4, GAGE, TRAILl, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3,
Legumain, Tie 3, PAGE4,VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET,
HER3, EPCAM, CA6, NAPI2B, TROP2, Claudin-6 (CLDN6), Claudin-16 (CLDN16),
CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, Uroplakin-1B (UPK1B), LIV1, ROR1, STRA6,
TMPRSS3, TMPRSS4, TMEM238, Clorf186, Fos-related antigen 1, VEGFR1, endoglin,
PD-
L1, VTCN1 (B7-H4), VISTA, or a fragment thereof, and a fragment thereof. In
some
embodiments, an amino acid sequence of the tumor antigen has at least 80%
sequence identity
with the amino acid sequence of a tumor antigen selected from TABLE 1. In some
embodiments,
an amino acid sequence of the tumor antigen has at least 80% sequence identity
with the amino
acid sequence of a tumor antigen selected from the group consisting of HER2,
EGFR, CMET,
HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII,
FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ES0-1,
Endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B,
STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, and LRRC15, but not HER2 when the
11
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
second binding domain specifically binds to CD40. In some embodiments, an
amino acid
sequence of the antigen on the antigen presenting cell has at least 80%
sequence identity with the
amino acid sequence of an antigen selected from the group consisting of CD40,
DEC-205, CD36
mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL,
CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A,
HVEM, PD-L1, CD32B, and CD47, but not CD40 when the first binding domain
specifically
binds to HER2. In some embodiments, an amino acid sequence of the antigen on
the antigen
presenting cell has at least 80% sequence identity with the amino acid
sequence of an antigen
selected from TABLE 2. In some embodiments, the second binding domain is a
CD40 agonist. In
some embodiments, the first binding domain comprises a single chain variable
fragment (scFv).
In some embodiments, the second binding domain is a single chain variable
fragment (scFv). In
some embodiments, the second binding domain comprises a single chain variable
fragment from
an anti-CD40 antibody, an anti-DEC-205 antibody, an anti-CD36 mannose
scavenger receptor 1
antibody, an anti-DC-SIGN antibody, an anti-CLEC9A antibody, an anti-CLEC12A
antibody, an
anti-BDCA-2 antibody, an anti-OX4OL antibody, an anti-41BBL antibody, an anti-
CD204
antibody, an anti-MARCO antibody, an anti-CLEC5A antibody, an anti-Dectin 1
antibody, an
anti-Dectin 2 antibody, an anti-CLEC10A antibody, an anti-CD206 antibody, an
anti-CD64
antibody, an anti-CD32A antibody, an anti-CD16A antibody, an anti-HVEM
antibody, an anti-
PD-L1, or an anti-CD32B antibody. In some embodiments, the second binding
domain is
attached to the Fc domain or the light chain of the first binding domain: a)
as an Fc domain-
second binding domain fusion peptide; b) as a light chain-second binding
domain fusion peptide;
or c) by a conjugation via a first linker. In some embodiments, the Fc domain
is attached to the
first binding domain: a) as an Fc domain-first binding domain fusion peptide;
or b) by
conjugation via a second linker. In some embodiments, the Fc domain is
attached to both the first
binding domain and to the second binding domain as a first binding domain-Fc
domain-second
binding domain fusion peptide. In some embodiments, the first binding domain
is attached to
both the Fc domain and the second binding domain as a first binding domain-
second binding
domain-Fc domain fusion peptide. In some embodiments, the first binding domain
and the Fc
domain comprise an antibody and the second binding domain comprises a single
chain variable
fragment (scFv). In some embodiments, the first binding domain has a set of
variable region
CDR sequences that comprises a set of variable region CDR sequences set forth
in TABLE 3 or
TABLE 4. In some embodiments, the second binding domain comprises a variable
domain
comprising a set of CDR sequences set forth in TABLE 11 or TABLE 12. In some
embodiments,
the first binding domain comprises a variable region comprising VH and VL
sequences at least
12
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
80% sequence identity to a pair of VH and VL sequences set forth in TABLE 5 or
TABLE 6. In
some embodiments, the second binding domain comprises a variable region having
VH and VL
sequences having at least 80% sequence identity to a VH or VL sequence set
forth in TABLE 13
or TABLE 14. In some embodiments, the first binding domain comprises an amino
acid sequence
having at least 80% sequence identity to any sequence in TABLE 7 or TABLE 8.
In some
embodiments, the second binding domain comprises an amino acid sequence having
at least 80%
sequence identity to any sequence in TABLE 15 or TABLE 16. In some
embodiments, the
second binding domain-Fc domain-first binding domain fusion peptide as
described herein
comprises an amino acid sequence having at least 80% sequence identity to a
sequence in
TABLE 9, TABLE 10, or TABLE 17. In some embodiments, the second binding domain-
first
binding domain-Fc domain fusion peptide as described herein comprises an amino
acid sequence
having at least 80% sequence identity to a sequence in TABLE 18 or TABLE 19.
[0018] In some aspects, a conjugate comprises: a) an immune-stimulatory
compound; b) an
antibody construct comprising a first binding domain and an Fc domain, wherein
the first binding
domain specifically binds to an antigen expressed on a cell, wherein the amino
acid sequence of
the antigen has at least 80% homology to the amino acid sequence of an antigen
selected from a
group consisting of Endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6,
CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, LRRC15, DEC-205,
CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, 41BBL,
MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A, CD16A, HVEM, and CD32B, and a
fragment thereof; and c) a linker attaching the antibody construct to the
immune-stimulatory
compound, wherein the linker is covalently bound to the antibody construct and
the linker is
covalently bound to the immune-stimulatory compound, and wherein a molar ratio
of immune-
stimulatory compound to antibody construct is less than 8.
[0019] In some aspects, a conjugate comprises: a) an immune-stimulatory
compound; b) an
antibody construct comprising a first binding domain and an Fc domain,
wherein: i) the first
binding domain specifically binds to an antigen, wherein the amino acid
sequence of the antigen
has at least 80% homology to the amino acid sequence of an antigen selected
from a group
consisting of endoglin, PD-L1, CD204, CD206, CD301, VTCN1, VISTA, GLP-3,
CLDN6,
CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, LRRC15, DEC-205,
CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL,
41BBL, MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A, CD16A, HVEM, CD32B, and
CD47, and a fragment thereof, ii) a Kd for binding of the first binding domain
to the antigen in a
presence of the immune-stimulatory compound is less than about 100 nM and no
greater than
13
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
about 100 times a Kd for binding of the first binding domain to the antigen in
the absence of the
immune-stimulatory compound, and iii) a Kd for binding of the Fc domain to an
Fc receptor in
the presence of the immune-stimulatory compound is no greater than about 100
times a Kd for
binding of the Fc domain to the Fc receptor in the absence of the immune-
stimulatory compound;
and c) a linker attaching the antibody construct to the immune-stimulatory
compound, wherein
the linker is covalently bound to the antibody construct and the linker is
covalently bound to the
immune-stimulatory compound, and wherein a molar ratio of immune-stimulatory
compound to
antibody construct is less than 8.
[0020] In some aspects, a conjugate comprises: a) an immune-stimulatory
compound; b) an
antibody construct comprising a first binding domain and an Fc domain,
wherein: i) the first
binding domain comprises a variable region comprising a set of CDR sequences
that comprises at
least 80% sequence identity to a set of variable region CDR sequences set
forth in TABLE 3 or
TABLE 11; ii) a Kd for binding of the first binding domain to the antigen in a
presence of the
immune-stimulatory compound is less than about 100 nM and no greater than
about 100 times a
Kd for binding of the first binding domain to the antigen in the absence of
the immune-
stimulatory compound, and iii) a Kd for binding of the Fc domain to an Fc
receptor in the
presence of the immune-stimulatory compound is no greater than about 100 times
a Kd for
binding of the Fc domain to the Fc receptor in the absence of the immune
stimulatory compound;
and c) a linker attaching the antibody construct to the immune-stimulatory
compound, wherein
the linker is covalently bound to the antibody construct and the linker is
covalently bound to the
immune-stimulatory compound, and wherein a molar ratio of immune-stimulatory
compound to
antibody construct is less than 8.
[0021] In some aspects, a conjugate for use in activating an immune cell
comprises: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein the first binding domain specifically binds to an
antigen expressed on a
cell, wherein the amino acid sequence of the antigen has at least 80% homology
to the amino
acid sequence of an antigen selected from a group consisting of Endoglin,
CD204, CD206,
CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4,
TMEM238, Clorf186, LRRC15, DEC-205, CD36 mannose scavenger receptor 1, CLEC9A,
DC-
SIGN, CLEC12A, BDCA-2, 41BBL, MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A,
CD16A, HVEM, and CD32B, and a fragment thereof; and c) a linker attaching the
antibody
construct to the immune-stimulatory compound, wherein the linker is covalently
bound to the
antibody construct and the linker is covalently bound to the immune-
stimulatory compound, and
wherein a molar ratio of immune-stimulatory compound to antibody construct is
less than 8; and
14
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
wherein immune cell activation caused by the conjugate when bound to the tumor
antigen as
measured by a cytokine release assay is greater than immune cell activation is
greater than
immune cell activation in the absence of binding to the tumor antigen.
[0022] In some aspects, a conjugate for use in activating an immune cell
comprises: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein: i) the first binding domain specifically binds to an
antigen, wherein the
amino acid sequence of the antigen has at least 80% homology to the amino acid
sequence of an
antigen selected from a group consisting of endoglin, PD-L1, CD204, CD206,
CD301, VTCN1,
VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238,
Clorf186, LRRC15, DEC-205, CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN,
CLEC12A, BDCA-2, OX4OL, 41BBL, MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A,
CD16A, HVEM, CD32B, and CD47, and a fragment thereof, ii) a Kd for binding of
the first
binding domain to the antigen in a presence of the immune-stimulatory compound
is less than
about 100 nM and no greater than about 100 times a Kd for binding of the first
binding domain to
the antigen in the absence of the immune-stimulatory compound, and iii) a Kd
for binding of the
Fc domain to an Fc receptor in the presence of the immune-stimulatory compound
is no greater
than about 100 times a Kd for binding of the Fc domain to the Fc receptor in
the absence of the
immune-stimulatory compound; and c) a linker attaching the antibody construct
to the immune-
stimulatory compound, wherein the linker is covalently bound to the antibody
construct and the
linker is covalently bound to the immune-stimulatory compound, and wherein a
molar ratio of
immune-stimulatory compound to antibody construct is less than 8; and wherein
immune cell
activation caused by the conjugate when bound to the tumor antigen as measured
by a cytokine
release assay is greater than immune cell activation is greater than immune
cell activation in the
absence of binding to the tumor antigen.
[0023] In some aspects, a conjugate for use in activating an immune cell
comprises: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein the first binding domain comprises a variable region
comprising a set of
CDR sequences that comprises at least 80% sequence identity to a set of
variable region CDR
sequences set forth in TABLE 3 or TABLE 11; c) a linker attaching the antibody
construct to the
immune-stimulatory compound, wherein the linker is covalently bound to the
antibody construct
and the linker is covalently bound to the immune-stimulatory compound, and
wherein a molar
ratio of immune-stimulatory compound to antibody construct is less than 8; and
wherein immune
cell activation caused by the conjugate when bound to the tumor antigen as
measured by a
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cytokine release assay is greater than immune cell activation is greater than
immune cell
activation in the absence of binding to the tumor antigen.
[0024] In some aspects, a conjugate for use in activating an immune cell
comprising: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein: i) the first binding domain comprises a variable region
comprising a set
of CDR sequences that comprises at least 80% sequence identity to a set of
variable region CDR
sequences set forth in TABLE 3 or TABLE 11; ii) a Kd for binding of the first
binding domain to
the antigen in a presence of the immune-stimulatory compound is less than
about 100 nM and no
greater than about 100 times a Kd for binding of the first binding domain to
the antigen in the
absence of the immune-stimulatory compound, and iii) a Kd for binding of the
Fc domain to an
Fc receptor in the presence of the immune-stimulatory compound is no greater
than about 100
times a Kd for binding of the Fc domain to the Fc receptor in the absence of
the immune
stimulatory compound; and c) a linker attaching the antibody construct to the
immune-
stimulatory compound, wherein the linker is covalently bound to the antibody
construct and the
linker is covalently bound to the immune-stimulatory compound, and wherein a
molar ratio of
immune-stimulatory compound to antibody construct is less than 8; and wherein
immune cell
activation caused by the conjugate when bound to the tumor antigen as measured
by a cytokine
release assay is greater than immune cell activation is greater than immune
cell activation in the
absence of binding to the tumor antigen. In some embodiments, the first
binding domain
comprises a variable region comprising VH and VL sequences at least 80%
sequence identity to a
pair of VH and VL sequences set forth in TABLE 5 or TABLE 13. In some
embodiments, the first
binding domain comprises an amino acid sequence having at least 80% sequence
identity to any
sequence in TABLE 7 or TABLE 15. In some embodiments, a Kd for binding of the
Fc domain
to the Fc receptor in the presence of the immune-stimulatory compound is no
greater than about
two times, five times, ten times, or fifty times a Kd for binding of the Fc
domain to the Fc
receptor in an absence of the immune-stimulatory compound. In some
embodiments, the
immune-stimulatory compound is a damage-associated molecular pattern molecule
or pathogen-
associated molecular pattern molecule. In some embodiments, the immune-
stimulatory
compound is a toll-like receptor agonist, STING agonist, or RIG-I agonist. In
some
embodiments, the immune-stimulatory compound is a CpG oligonucleotide, Poly
G10, Poly G3,
Poly I:C, Lipopolysaccharide, zymosan, flagellin, Pam3CSK4, PamCysPamSK4,
dsRNA, a
diacylated lipopeptide, a triacylated lipoprotein, lipoteichoic acid, a
peptidoglycan, a cyclic
dinucleotide, a 5'ppp-dsRNA, S-27609, CL307, UC-IV150, imiquimod, gardiquimod,
resiquimod, motolimod, VTS-1463G5-9620, G5K2245035, TMX-101, TMX-201, TMX-202,
16
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240,
KU34B,
SB9200, SB11285, 8-substituted imidazo[1,5-a]pyridine, or CL663. In some
embodiments, the
immune-stimulatory compound is an inhibitor of TGFB, Beta-Catenin, TNIK,
Tankyrase, PI3K-
beta, STAT3, IL-10, IDO, or TDO. In some embodiments, the immune-stimulatory
compound is
LY2109761, G5K263771, iCRT3, iCRT5, iCRT14, LY2090314, CGX-1321, PRI-724,
BC21,
ZINCO2092166, LGK974, IWP2, LY3022859, LY364947, 5B431542, AZD8186, SD-208,
indoximod (NLG8189), F001287, GDC-0919, epacadostat (INCB024360), RG70099, 1-
methyl-
L-tryptophan, methylthiohydantoin tryptophan, brassinin, annulin B, exiguamine
A, PIM, LM10,
8-substituted 2-amino-3H-benzo[b]azepine-4-carboxamide, or INCB023843. In some
embodiments, the Fc domain is an Fc domain variant comprising at least one
amino acid residue
change as compared to a wild type sequence of the Fc domain. In some
embodiments, the Fc
domain variant binds to an Fc receptor with altered affinity as compared to
the wild type Fc
domain. In some embodiments, the at least one amino acid residue change is
selected from a
group consisting of: a) F243L, R292P, Y300L, L235V, and P396L, wherein
numbering of amino
acid residues in the Fc domain is according to the EU index; b) 5239D and
I332E, wherein
numbering of amino acid residues in the Fc domain is according to the EU
index; and c) 5298A,
E333A, and K334A, wherein numbering of amino acid residues in the Fc domain is
according to
the EU index. In some embodiments, the antibody construct or conjugate induces
secretion of
cytokines by an immune cell as measured by a cytokine release assay. In some
embodiments, the
cytokine is IFN-y, IL-8, IL-12, IL-2, or a combination thereof. In some
embodiments, the
antibody construct or conjugate induces antigen presentation on a dendritic
cell, B cell,
macrophage, or a combination thereof.
[0025] In some aspects, a method of making a conjugate comprises linking an
antibody
construct as described herein to an immune stimulatory compound by a linker.
[0026] In some aspects, a pharmaceutical composition comprises the conjugate
or antibody
construct of as described herein and a pharmaceutically acceptable carrier.
[0027] In some aspects, a method of treatment for a subject in need thereof
comprises
administering a therapeutic dose of the antibody construct or conjugate as
described herein or the
pharmaceutical composition as described herein. In some embodiments, the
subject has cancer.
In some embodiments, the antibody construct or conjugate is administered
intravenously,
cutaneously, subcutaneously, or injected at a site of affliction. In some
embodiments, after
administration of antibody construct or conjugate to the subject, immune cell
activation is
increased in the subject as measured by a secretion of one or more cytokines
as measured by a
cytokine release assay, a secretion of one or more chemokines as measured by
an ELISA
17
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
immunoassay, an expression level of one or more cell surface proteins
associated with immune
stimulation as measured by an ELISA immunoassay, an activity of one or more
immune cell
functions, or combination thereof, as compared to before administration of the
antibody construct
or conjugate to the subject. In some embodiments, the activity of one or more
immune cell
functions comprises antibody-dependent cell-mediated cytotoxicity as measured
by an ADCC
assay, antibody dependent cellular phagocytosis as measured by an ADCP assay,
or antigen
cross-presentation as measured by a cross-presentation assay. In some
embodiments, after
administration of the antibody construct or conjugate to the subject, tumor
cell intracellular
signaling is altered in the subject as compared to tumor cell intracellular
signaling before
administration of the antibody construct or conjugate as measured by an
intracellular signaling
assay. In some embodiments, the altered tumor cell intracellular signaling
increases tumor
immunogenicity as measured by an immunogencity assay.
[0028] In some aspects, a kit comprising a pharmaceutically acceptable dosage
unit of a
pharmaceutically effective amount of the conjugate or antibody construct as
described herein or
the pharmaceutical composition as described herein.
[0029] Described herein are recombinant bispecific antibodies useful in the
treatment of cancer.
The recombinant antibodies according to the current disclosure are bispecific
antibodies that can
comprise at least two different antigen binding domains that are coupled to an
Fc comprising
domain. This recombinant antibody can exhibit more potent immune activation
when both
antigen binding domains are bound to their respective antigen. One example
method for
increasing immune activation when both antigen binding domains are bound to
their respective
antigen can be accomplished by a recombinant antibody coupled to an Fc
comprising domain that
exhibits reduced affinity to an Fc receptor. Another example method for
achieving an increased
immune activation when both antigen binding domains are bound to their
respective antigen can
be accomplished by using a binding domain with a low avidity for its antigen
as one of the
antigen binding domains in the recombinant antibody. One binding domain of the
bispecific
antibody can specifically bind to a tumor associated antigen and another
binding domain can
specifically bind to a molecule on the surface of an antigen presenting cell
(APC), such as a
macrophage or dendritic cell. Thus, the two binding domains cooperate to bring
APCs to
cancerous cells or tumors allowing the APC to initiate/propagate a cancer
cell/tumor specific
immune response through cytokine release, chemokine release, or presentation
of tumor
associated antigens to effector or helper T cells.
[0030] Therapy with recombinant monoclonal or bispecific antibodies can
generally be well
tolerated, however, antibodies directed to immune response stimulating
receptors on immune
18
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cells can result in systemic toxic release of cytokines and other immune
modulators that can limit
their clinical use or dose, thereby limiting their effectiveness in generating
patient anti-tumor
responses. This immune activation can be especially non-beneficial when it
occurs systemically
in the absence of tumor antigens. The systemic agonism exhibited by antibodies
to many APC
receptors can depend upon high affinity binding to the APC antigen and higher
order cross-
linking of the APC receptors by clustering of the cell bound antibodies. Many
studies show this
can be mediated by Fc gamma Receptor (FcyR) binding to the Fc domain of the
antibodies, and
cross-linking of different antibody molecules and their bound APC immune
stimulating
receptors. An additional complication of cross-linking by FcyR can be antibody
dependent cell
mediated cytotoxicity (ADCC) of the APCs resulting in lowered immune response
to tumors and
pathogens. ADCC can be attributed to the antibody Fc region which binds to
FcyRs on effector
cells (e.g., NK cells). Two non-mutually exclusive solutions to the above can
be contemplated. In
one, elevating the threshold for FcyR binding can reduce excessive systemic
immune activation
and unwanted ADCC directed to APCs of antibody therapy. In the second, the
affinity of the
antibody for its APC target can be lowered so that effective agonistic binding
of antibody
molecules to APCs can be driven by avidity, preferentially found when the
bispecific antibody is
bound to its tumor antigen target. As described herein, the Fc comprising
region of the
recombinant bispecific antibody can contain one or more mutations that can
reduce binding to an
FcyR. Alternatively, the Fc region can be derived from an IgG subclass that
can bind FcyRs with
low affinity, for example IgG2. Fc receptors can be highly expressed on
different antigen
presenting cells such as dendritic cells, and their engagement can lead to
activation of the
immunostimulatory and antigen presenting function of these cells. By reducing
binding of the Fc
region to the FcyR the threshold for APC activation can be raised. By raising
the threshold for
APC activation, the possibility of a damaging immune/inflammatory response to
healthy, non-
cancerous tissue can be reduced. Attenuating activation by modifications made
to the Fc regions
can result in superior bioavailability and lower side effects. Also described
herein are bispecific
antibodies with high affinity anti-tumor antigen binding and low affinity
immune receptor
binding such that APC activation can be increased when the bispecific antibody
is bound to its
tumor antigen. As a result, the antibodies of this disclosure generally can
have a higher maximum
tolerated dosage, and can be administered at levels higher than therapeutic
antibodies not
modified as described herein.
[0031] In some embodiments, the recombinant bispecific antibody further
comprises a
chemotherapeutic compound and a linker, wherein the linker links the
chemotherapeutic
compound to the Fc comprising domain. In some aspects, the chemotherapeutic
compound
19
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprises an alkylating agent, an anthracycline, a cytoskeletal disruptor, a
histone deacetylase
inhibitor, an inhibitor of, a kinase inhibitor, a nucleoside analog or
precursor analog, a peptide
antibiotic, a platinum-based compound, or a plant alkaloid.
[0032] In some aspects, the recombinant bispecific antibody specifically binds
to the tumor
associated antigen in a cluster of recombinant antibodies and induces a signal
in the antigen
presenting cell. In some aspects, the recombinant antibody specifically binds
to the tumor
associated antigen in a cluster of recombinant antibodies and results in an
increased avidity for
the molecule on the antigen presenting cell. In some aspects, a recombinant
antibody density
resulting from the recombinant antibody binding to the tumor associated
antigen induces
signaling in the antigen presenting cell. In some aspects, the recombinant
antibody density of
greater than 5000 antibodies per cell resulting from the recombinant antibody
specifically
binding to the tumor associated antigen induces signaling in the antigen
presenting cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The novel features of the disclosure are set forth with particularity
in the appended
claims. A better understanding of the features and advantages of the present
disclosure will be
obtained by reference to the following detailed description that sets forth
illustrative aspects, in
which the principles of the disclosure are utilized, and the accompanying
drawings of which:
[0034] FIGURE 1 illustrates a schematic of an antibody construct comprising an
antibody and
a second binding domain. An antibody can comprise two heavy chains as shown in
gray and two
light chains as shown in light gray. A portion of the heavy chains can
comprise Fc domains (705
and 720). An antibody can comprise a binding domain comprising two antigen
binding sites (710
and 715). The second binding domain can be attached to the antibody (780 and
785), for
example, at the C-terminus of the heavy chains.
[0035] FIGURE 2 illustrates a schematic of an exemplary conjugate. A conjugate
can comprise
an antibody, which can comprise two heavy chains as shown in gray and two
light chains as
shown in light gray. The antibody can comprise a binding domain comprising two
antigen
binding sites (910 and 915), and a portion of the heavy chains contain Fc
domains (905 and 920).
The immune-stimulatory compounds (930 and 940) can be conjugated to the
antibody by linkers
(960 and 970). A second binding domain can be attached to the antibody (980
and 985), for
example, at the C-terminus of the heavy chains.
[0036] FIGURE 3 illustrates a schematic of an exemplary conjugate. A conjugate
can comprise
the Fc region of an antibody with the heavy chains shown in gray, and two
scaffolds as shown in
light gray. The conjugate can comprise a first binding domain comprising two
antigen binding
sites (1110 and 1115) in the scaffolds, and a portion of the heavy chains can
comprise Fc
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
domains (1105 and 1120). The immune-stimulatory compounds (1130 and 1140) can
be
conjugated to the scaffolds by linkers (1160 and 1170). A second binding
domain can be attached
to the conjugate (1180 and 1185), for example, at the C-terminus of the heavy
chains.
[0037] FIGURE 4 illustrates a schematic of an exemplary conjugate. A conjugate
can comprise
the F(ab')2 region of an antibody with heavy chains shown in gray and light
chains shown in light
gray, and two scaffolds as shown in dark gray. The conjugate can comprise a
first binding
domain comprising two antigen binding sites (1310 and 1315), and a portion of
two scaffolds
contain Fc domains (1340 and 1345). The immune-stimulatory compounds (1330 and
1340) can
be conjugated to the scaffold by linkers (1360 and 1370). A second binding
domain can be
attached to the conjugate (1380 and 1385).
[0038] FIGURE 5 illustrates a schematic of an exemplary conjugate. A conjugate
can comprise
two scaffolds as shown in light gray and two scaffolds as shown in dark gray.
The conjugate can
comprise a first binding domain comprising two antigen binding sites (1510 and
1515), and a
portion of the two dark gray scaffolds contain Fc domains (1540 and 1545). The
immune-
stimulatory compounds (1530 and 1535) can be conjugated to the scaffolds by
linkers (1560 and
1570). A second binding domain can be attached to the conjugate (1580 and
1585).
[0039] FIGURE 6 illustrates a CLUSTAL 0(1.2.1) multiple amino acid sequence
alignment of
the amino acid sequences of SBT-040-G1VLPLL (SEQ ID NO: 1323), SBT-040-G1AAA
(SEQ
ID NO: 1324), SBT-040-G1WT (SEQ ID NO: 1325), and SBT-040-G1DE (SEQ ID NO:
1326).
The SBT-040-G1VLPLL sequence is an amino acid sequence of an IgG1 isotype
heavy chain of
a human CD40 monoclonal antibody SBT-040 containing L235V, F243L, R292P,
Y300L, and
P396L amino acid residue modifications of a wild type IgG1 Fc domain. The
L235V, F243L,
R292P, Y300L, and P396L amino acid residue modifications are in bold. The SBT-
040-G1AAA
sequence is an amino acid sequence of an IgG1 isotype heavy chain of a human
CD40
monoclonal antibody SBT-040 containing 5298A, E333A, and K334A amino acid
residue
modifications of a wild type IgG1 Fc domain. The 5298A, E333A, and K334A amino
acid
residue modifications are italics. The SBT-040-G1WT sequence is an amino acid
sequence of an
IgG1 isotype heavy chain of a human CD40 monoclonal antibody SBT-040. The SBT-
040-
G1AAA sequence is an amino acid sequence of an IgG1 isotype heavy chain of a
human CD40
monoclonal antibody SBT-040 containing 5239D and I332E amino acid residue
modifications
bold italics. Additionally, the hinge region of each amino acid sequence is
differentiated from
other regions of the amino acid sequence by brackets. The left bracket
indicates the upper portion
of the hinge region (UH). The four residues between the brackets are the
middle portion of the
hinge region. The right bracket indicates the lower portion of the hinge
region (LH). SEQ ID NO:
21
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
1327 is the sequence of SBT-040-G1VLPLL without the leader sequence. SEQ ID
NO: 1328 is
the sequence of SBT-040-G1AAA without the leader sequence. SEQ ID NO: 577 is
the sequence
of SBT-040-G1WT without the leader sequence. SEQ ID NO: 1329 is the sequence
of SBT-040-
GIDE without the leader sequence.
[0040] FIGURES 7A and 7B illustrate that a bispecific anti-HER2 x anti-CD40
IgG1
conjugate (HER2-CD40G1) and a bispecific anti-HER2 x anti-CD40 IgG1 Fc null
antibody
(HER2-CD40 Glnull) had decreased binding to CD40 on monocyte-derived dendritic
cells
(moDCs) compared to the parental anti-CD40 monoclonal antibody (SBT-040G1).
moDCs were
stained with either SBT-040G1, HER2-CD40G1 and HER2-CD40 Glnun at equivalent
molar
concentrations. A secondary goat anti-human IgG polyclonal antibody was used
to detect SBT-
040G1, HER2-CD40G1 or HER2-CD40 Glnun binding by flow cytometry. MFI fold
change was
calculated as (MFI test Ab/MFI isotype control).
[0041] FIGURE 8A illustrates activation of dendritic cells (DCs) was dependent
on CD40
agonism and Fc receptor agonism by bispecific anti-HER2-anti-CD40 IgG1
antibody construct
(HER2-CD40G1) bound to the tumor antigen HER2 as shown by increased expression
of CD86.
This figure also illustrates the anti-HER2 x anti-CD40 IgG1 Fc null antibody
(HER2-CD40
Glnun) conditional activation of dendritic cells (DCs) when bound to the tumor
antigen HER2.
CD86 was measured by flow cytometry on DCs co-cultured with CHO cells with or
without
HER2 expression in the presence of the HER2-CD40G1 antibody construct, anti-
HER2-anti-
CD40 IgG1 Fc null (HER2-CD4OG1nun), or the parental anti-CD40 monoclonal
antibody (SBT-
040G1) at the indicated concentrations. Each data point was generated from
pooled duplicate
samples. HER2 CHO indicates co-culture with HER2 expressing CHO cells; HERT
CHO
indicates a co-culture with CHO cells that were not expressing HER2.
[0042] FIGURE 8B illustrates activation of dendritic cells (DCs) was dependent
on CD40
agonism and Fc receptor agonism by bispecific anti-HER2-anti-CD40 IgG1
antibody construct
(HER2-CD40G1) bound to the tumor antigen HER2 as shown by increased expression
of CD83.
This figure also illustrates anti-HER2 x anti-CD40 IgG1 Fcnull (HER2-
CD40G1null) antibody
conditional activation of dendritic cells (DCs) when bound to the tumor
antigen HER2. CD83
was measured by flow cytometry on DCs co-cultured with CHO cells with or
without HER2
expression in the presence of the HER2-CD40G1 antibody construct, anti-HER2 x
anti-CD40
IgG1 Fcnun (HER2-CD4OG1nun), or the parental anti-CD40 monoclonal antibody
(SBT-040G1) at
the indicated concentrations. Each data point was generated from pooled
duplicate samples.
HER2 ' CHO indicates co-culture with HER2 expressing CHO cells; HERT CHO
indicates a co-
culture with CHO cells that were not expressing HER2.
22
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0043] FIGURE 9 illustrates macrophage-mediated antibody-dependent cellular
cytotoxicity
(ADCC) of HERZ target cells was efficiently induced by bispecific anti-HER2 x
anti-CD40
IgG1 antibody construct (HER2-CD40G1). Monocyte-derived macrophages were
generated by
culturing monocytes for 7 days in the presence of GM-CSF. Macrophages were
plated with
HER2-expressing CHO cells at a 2:1 ratio in the presence of titrating
concentrations of HER2-
CD40G1 antibody construct, anti-HER2-anti-CD40 IgG1 Fc null antibody construct
(HER2-
CD40G1nu11), parental anti-CD40 monoclonal antibody (SBT-040G1), or parental
anti-HER2
monoclonal antibody (SBT-050G1). After 24 hours, CHO viability was assessed by
flow
cytometry as a readout of ADCC activity.
[0044] FIGURE 10 illustrates schematics for three separate non-limiting
embodiments of
recombinant bispecific antibodies.
[0045] FIGURE 11 illustrates a schematic of an antibody construct comprising
an antibody and
a second binding domain. An antibody can comprise two heavy chains as shown in
gray and two
light chains as shown in light gray. A portion of the heavy chains can
comprise Fc domains (1705
and 1720). An antibody can comprise a binding domain comprising two antigen
binding sites
(1710 and 1715). The second binding domain can be attached to the antibody
(1780 and 1785),
for example, at the C-terminus of the light chains.
[0046] FIGURE 12 illustrates a schematic of an exemplary conjugate. A
conjugate can
comprise an antibody, which contains two heavy chains as shown in gray and two
light chains as
shown in light gray. The antibody can comprise a binding domain comprising two
antigen
binding sites (1910 and 1915), and a portion of the heavy chains can comprise
Fc domains (1905
and 1920). The immune-stimulatory compounds (1930 and 1940) can be conjugated
to the
antibody by linkers (1960 and 1970). A second binding domain can be attached
to the antibody
(1980 and 1985), for example, at the C-terminus of the light chains.
[0047] FIGURE 13 illustrates a schematic of an exemplary conjugate. A
conjugate can
comprise the Fc region of an antibody shown in gray, and two scaffolds as
shown in light gray.
The conjugate can comprise a first binding domain comprising two antigen
binding sites (2110
and 2115) in the scaffolds, and a portion containing Fc domains (2105 and
2120). The immune-
stimulatory compounds (2130 and 2140) can be conjugated to the scaffolds by
linkers (2160 and
2170). A second binding domain can be attached to the conjugate (2180 and
2185).
[0048] FIGURE 14 illustrates a schematic of an exemplary conjugate. A
conjugate can
comprise the F(ab')2 region of an antibody with heavy chains shown in gray and
light chains
shown in light gray, and two scaffolds as shown in dark gray. The conjugate
can comprise a first
binding domain comprising two antigen binding sites (2310 and 2315), and a
portion of two
23
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
scaffolds can comprise Fc domains (2340 and 2345). The immune-stimulatory
compounds (2330
and 2340) can be attached to the scaffolds by linkers (2360 and 2370). A
second binding domain
can be attached to the conjugate (2380 and 2385), for example, at the C-
terminus of the light
chains.
[0049] FIGURE 15 illustrates a schematic of an exemplary conjugate. A
conjugate can
comprise two scaffolds as shown in light gray and two scaffolds as shown in
dark gray. The
conjugate can comprise a first binding domain comprising two antigen binding
sites (2510 and
2515), and a portion of the two dark gray scaffolds contain Fc domains (2540
and 2545). The
immune-stimulatory compounds (2530 and 2540) can be attached to the scaffolds
by linkers
(2560 and 2570). A second binding domain can be attached to the conjugate
(2580 and 2585).
[0050] FIGURE 16 illustrates a schematic of an antibody construct comprising
an antibody. An
antibody can comprise two heavy chains and two light chains. A portion of the
heavy chains can
comprise Fc domains (2705 and 2720). An antibody can comprise a binding domain
comprising
two antigen binding sites shown in black (2710 and 2715).
[0051] FIGURE 17 illustrates a schematic of an antibody construct comprising
an antibody. An
antibody can comprise two heavy chains and two light chains. A portion of the
heavy chains can
comprise Fc domains (2925 and 2930). An antibody can comprise a first binding
domain
comprising two antigen binding sites shown in black (2910 and 2915). An
antibody can comprise
a second binding domain comprising two single chain variable fragments (2905
and 2920)
attached to a C-terminus of the light chains. A single chain variable fragment
can be attached to a
light chain chain at a heavy chain variable domain of the single chain
variable fragment. A single
chain variable fragment can be attached to a light chain at a light chain
variable domain of the
single chain variable fragment.
[0052] FIGURE 18 illustrates a schematic of an antibody construct comprising
an antibody. An
antibody can comprise two heavy chains and two light chains. A portion of the
heavy chains can
comprise Fc domains (3120 and 3125). An antibody can comprise a first binding
domain
comprising two antigen binding sites shown in black (3110 and 3115). An
antibody can comprise
a second binding domain comprising two single chain variable fragments (3130
and 3135)
attached to a C-terminus of the heavy chains. A single chain variable fragment
can be attached to
a heavy chain chain at a heavy chain variable domain of the single chain
variable fragment. A
single chain variable fragment can be attached to a heavy chain at a light
chain variable domain
of the single chain variable fragment.
[0053] FIGURE 19 illustrates a schematic of an antibody construct comprising
an antibody. An
antibody can comprise two heavy chains and two light chains. A portion of the
heavy chains can
24
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprise Fc domains (3330 and 3335). An antibody can comprise a first binding
domain
comprising two antigen binding sites shown in black (3310 and 3315). An
antibody can comprise
a second binding domain comprising two single chain variable fragments (3320
and 3325)
attached to a C-terminus of the light chains. A single chain variable fragment
can be attached to a
light chain chain at a heavy chain variable domain of the single chain
variable fragment. A single
chain variable fragment can be attached to a light chain at a light chain
variable domain of the
single chain variable fragment. An antibody can comprise a third binding
domain comprising two
single chain variable fragments (3340 and 3345) attached to a C-terminus of
the heavy chains. A
single chain variable fragment can be attached to a heavy chain chain at a
heavy chain variable
domain of the single chain variable fragment. A single chain variable fragment
can be attached to
a heavy chain at a light chain variable domain of the single chain variable
fragment.
[0054] FIGURE 20 shows that HER2-TLR8 agonist conjugates and HER2 x CD40 TLR8
agonist conjugates were active in the presence of PBMCs and SKBR3 cells that
express HER2,
as measured by TNFa production. HER2 antibody is HER2-G1WT.
[0055] FIGURE 21 shows that TROP2(TROP2-G1WT)-TLR8 agonist conjugates were
active
in the presence of PBMCs and SKBR3 cells that express HER2, as measured by
TNFa
production. TROP2 antibody is TROP2-G1WT.
[0056] FIGURE 22 shows that a CEA -TLR8 agonist conjugate was active in the
presence of
monocytes and CHO cells engineered to express CEA, while the CEA antibody
alone, and the
control antibodies (HER2-G1WT) and conjugates were not active, as measured by
TNFa
production. CEA antibody is CEA-G1WT.
[0057] FIGURE 23 shows that an anti-CEA-TLR8 agonist conjugate and a CEA x
CD40 TLR8
agonist conjugate were active in the presence of monocytes and SKCO-1 cells,
as measured by
TNFa production. CEA antibody is CEA-G1WT and bispecific CEA x CD40 antibody
is CEA x
CD4O-G1WT.
[0058] FIGURE 24 shows that a TROP TRL8 agonist conjugate was active in a dose-
dependent manner on various cell lines expressing TROP2.
[0059] FIGURE 25 shows that a TROP2 TLR8 agonist conjugate was active in a
dose-
dependent manner on various cell lines expressing TROP2.
[0060] FIGURE 26 shows that a HER2 x CD40 bispecific antibody conjugate was
able to
activate monocyte-derived dendritic cells.
[0061] FIGURE 27 shows that a HER2 x CD40 bispecific antibody conjugate was
further able
to stimulate T cells in the presence of HER2 postive tumor cells.
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0062] FIGURES 28A, 28B, and 28C show that activation of primary B cells (CD86
expression) was increased by bispecific HER2 x CD40 recombinant antibody
conjugate as
compared to a Her2 recombinant antibody conjugate.
DETAILED DESCRIPTION
[0063] Additional aspects and advantages of the present disclosure will become
apparent to
those skilled in this art from the following detailed description, wherein
illustrative aspects of the
present disclosure are shown and described. As will be realized, the present
disclosure is capable
of other and different aspects, and its several details are capable of
modifications in various
respects, all without departing from the disclosure. Accordingly, the drawings
and description are
to be regarded as illustrative in nature, and not as restrictive.
[0064] As used herein, "homologous" or "homology" refers to the similarity
between a DNA,
RNA, nucleotide, amino acid, or protein sequence to another DNA, RNA,
nucleotide, amino
acid, or protein sequence. Homology can be expressed in terms of a percentage
of sequence
identity of a first sequence to a second sequence. Percent (%) sequence
identity with respect to a
reference DNA sequence can be the percentage of DNA nucleotides in a candidate
sequence that
are identical with the DNA nucleotides in the reference DNA sequence after
aligning the
sequences. Percent (%) sequence identity with respect to a reference amino
acid sequence can be
the percentage of amino acid residues in a candidate sequence that are
identical with the amino
acid residues in the reference amino acid sequence after aligning the
sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence identity, and not
considering any
conservative substitutions as part of the sequence identity.
[0065] As used herein, the term "antibody" refers to an immunoglobulin
molecule that
specifically binds to, or is immunologically reactive toward, a specific
antigen. Antibody can
include, for example, polyclonal, monoclonal, genetically engineered, and
antigen binding
fragments thereof. An antibody can be, for example, murine, chimeric,
humanized,
heteroconjugate, bispecific, diabody, triabody, or tetrabody. The antigen
binding fragment can
include, for example, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAbs (heavy chain
antibodies), a single
domain antibody, VHH, VNAR, sdAbs, or nanobody.
[0066] As used herein a "recombinant antibody" is an antibody that comprises
an amino acid
sequence derived from two different species or, or two different sources, and
includes synthetic
molecules. By way of non-limiting example, an antibody that comprises a non-
human CDR and a
human variable region framework or constant or Fc region, an antibody with
binding domains
from two different monoclonal antibodies, or an antibody comprising a mutation
of one or more
amino acid residues to increase or decrease biological activity or binding of
a part of the
26
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
antibody. In certain embodiments, recombinant antibodies are produced from a
recombinant
DNA molecule or synthesized. In certain embodiments, the antibodies described
herein are a
polypeptide(s) encoded by one or more polynucleotides.
[0067] As used herein, "recognize" refers to the association or binding
between an antigen
binding domain and an antigen.
As used herein, an "antigen" refers to an antigenic substance that can trigger
an immune response
in a host. An antigenic substance can be a molecule, such as a costimulatory
molecule (e.g.,
CD40, OX4OL, 4-1BBL, DEC-205, etc.) that can trigger an immune response in a
host.
[0068] As used herein, a "tumor antigen" refers to an antigenic substance
associated with a
tumor or cancer cell, and that can trigger an immune response in a host.
[0069] As used herein, an "antigen on an antigen presenting cell" refers to an
antigenic
substance associated with an antigen presenting, and that can trigger an
immune response in a
host.
[0070] As used herein, an "antibody construct" refers to a construct that
contains an antigen
binding domain and an Fc domain.
[0071] As used herein, a binding domain refers to an antibody or non-antibody
domain.
[0072] As used herein, an "antigen binding domain" refers to a binding domain
from an
antibody or from a non-antibody that can bind to an antigen. An antigen
binding domain can be a
tumor antigen binding domain or a binding domain that can bind to an antigen
(such as a
molecule) on an antigen presenting cell. Antigen binding domains can be
numbered when there is
more than one antigen binding domain in a given conjugate or antibody
construct (e.g., first
antigen binding domain, second antigen binding domain, third antigen binding
domain, etc.).
Different antigen binding domains in the same conjugate or construct can
target the same antigen
or different antigens (e.g., first antigen binding domain that can bind to a
tumor antigen, second
antigen binding domain that can bind to a molecule on an antigen presenting
cell (APC antigen),
and third antigen binding domain that can bind to an APC antigen).
[0073] As used herein, an "antibody antigen binding domain" refers to a
binding domain from
an antibody that can bind to an antigen.
[0074] As used herein, an "Fe domain" refers to an Fc domain from an antibody
or from a non-
antibody that can bind to an Fc receptor. As used herein, an "Fe domain" and
an "Fe comprising
domain" can be used interchangeably.
[0075] As used herein, a "target binding domain" refers to a construct that
contains an antigen
binding domain from an antibody or from a non-antibody that can bind to an
antigen.
27
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0076] As used herein, an "ATAC" refers to a construct of an immune-
stimulatory compound
and a linker.
[0077] As used herein, a "conjugate" refers to an antibody construct attached
to an immune-
stimulatory molecule.
[0078] As used herein, a "bispecific tumor targeting antibody construct"
refers to a structure that
comprises a tumor antigen binding domain, a binding domain that binds to a
molecule on an
immune cell, such as an antigen presenting cell, and an Fc domain. A
bispecific tumor targeting
conjugate refers to bispecific tumor targeting antibody construct attached to
an immune-
stimulatory compound. As used herein, a "bispecific tumor targeting antibody
construct" is used
interchangeably with a "recombinant bispecific antibody". As used herein, a
"bispecific tumor
targeting antibody conjugate" is used interchangeably with a "recombinant
bispecific antibody
conjugate".
[0079] As used herein, an "immune cell" refers to a T cell, B cell, NK cell,
NKT cell, or an
antigen presenting cell. In some embodiments, an immune cell is a T cell, B
cell, NK cell, or
NKT cell. In some embodiments, an immune cell is an antigen presenting cell.
In some
embodiments, an immune cell is not an antigen presenting cell.
[0080] As used herein, "minimum anticipated biological effect level" (MABEL)
is the
anticipated dose needed that results in a biological effect in a human
subject, in which a
biological effect is measured by an in vitro, ex vivo, and/or in vivo assay
that measures a selected
biological, biochemical, pharmacological, or pharmacodynamic effect. A
selected biological,
biochemical, pharmacological, or pharmacodynamic effect can be secretion of
one or more
cytokines, secretion of one or more chemokines, expression level of one or
more cell surface
proteins associated with immune stimulation, or activity of one or more immune
cell functions.
Cytokine release can be measured by a cytokine release assay. Chemokine
secretion can be
measured by an ELISA immunoassay. Expression level of one or more cell surface
proteins
associated with immune stimulation can be measured by Fluorescent-Activated
Cell Sorting
(FACS). Activity of one or more immune cell functions can be antibody-
dependent cell-mediated
cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or
antigen cross-
presentation. ADCC can be measured by an ADCC assay. ADCP can be measured by
an ACDP
assay. Antigen cross-presentation can be measured by a cross-presentation
assay.
[0081] As used herein, the abbreviations for the natural 1-enantiomeric amino
acids are
conventional and can be as follows: alanine (A, Ala); arginine (R, Arg);
asparagine (N, Asn);
aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine
(Q, Gln); glycine (G,
Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K,
Lys); methionine (M,
28
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T,
Thr); tryptophan (W,
Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X can
indicate any amino
acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H),
lysine (K), or
arginine (R).
[0082] The term "salt" or "pharmaceutically acceptable salt" refers to salts
derived from a
variety of organic and inorganic counter ions well known in the art.
Pharmaceutically acceptable
acid addition salts can be formed with inorganic acids and organic acids.
Inorganic acids from
which salts can be derived include, for example, hydrochloric acid,
hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, and the like. Organic acids from which
salts can be derived
include, for example, acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,
benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic acid, salicylic
acid, and the like. Pharmaceutically acceptable base addition salts can be
formed with inorganic
and organic bases. Inorganic bases from which salts can be derived include,
for example, sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum,
and the like. Organic bases from which salts can be derived include, for
example, primary,
secondary, and tertiary amines, substituted amines including naturally
occurring substituted
amines, cyclic amines, basic ion exchange resins, and the like, specifically
such as
isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine,
and ethanolamine.
In some embodiments, the pharmaceutically acceptable base addition salt is
chosen from
ammonium, potassium, sodium, calcium, and magnesium salts.
[0083] The term "Cx_y" when used in conjunction with a chemical moiety, such
as alkyl, alkenyl,
or alkynyl is meant to include groups that contain from x to y carbons in the
chain. For example,
the term "Cx_yalkyl" refers to substituted or unsubstituted saturated
hydrocarbon groups,
including straight-chain alkyl and branched-chain alkyl groups that contain
from x to y carbons
in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-
trifluoroethyl, etc.
[0084] The terms "Cx_yalkenyl" and "Cx_yalkynyl" refer to substituted or
unsubstituted
unsaturated aliphatic groups analogous in length and possible substitution to
the alkyls described
above, but that contain at least one double or triple bond respectively.
[0085] The term "carbocycle" as used herein refers to a saturated, unsaturated
or aromatic ring in
which each atom of the ring is carbon. Carbocycle includes 3- to 10-membered
monocyclic rings,
6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each
ring of a bicyclic
carbocycle may be selected from saturated, unsaturated, and aromatic rings. In
an exemplary
embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or
unsaturated ring, e.g.,
29
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated,
unsaturated and
aromatic bicyclic rings, as valence permits, is included in the definition of
carbocyclic.
Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl,
adamantyl, phenyl,
indanyl, and naphthyl.
[0086] The term "heterocycle" as used herein refers to a saturated,
unsaturated or aromatic ring
comprising one or more heteroatoms. Exemplary heteroatoms include N, 0, Si, P,
B, and S
atoms. Heterocycles include 3- to 10-membered monocyclic rings, 6- to 12-
membered bicyclic
rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic
heterocycle may be selected
from saturated, unsaturated, and aromatic rings wherein at least one of the
rings includes a
heteroatom. In an exemplary embodiment, an aromatic ring, e.g., pyridyl, may
be fused to a
saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, morpholine,
piperidine or
cyclohexene. The term "heteroaryl" includes aromatic single ring structures,
preferably 5- to 7-
membered rings, more preferably 5- to 6-membered rings, whose ring structures
include at least
one heteroatom, preferably one to four heteroatoms, more preferably one or two
heteroatoms.
The term "heteroaryl" also include polycyclic ring systems having two or more
cyclic rings in
which two or more carbons are common to two adjoining rings wherein at least
one of the rings
is heteroaromatic, e.g., the other cyclic rings can be aromatic or non-
aromatic carbocyclic, or
heterocyclic. Heteroaryl groups include, for example, pyrrole, furan,
thiophene, imidazole,
oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine,
and the like.
[0087] The term "substituted" refers to moieties having substituents replacing
a hydrogen on one
or more carbons or substitutable heteroatoms, e.g., NH, of the structure. It
will be understood that
"substitution" or "substituted with" includes the implicit proviso that such
substitution is in
accordance with permitted valence of the substituted atom and the substituent,
and that the
substitution results in a stable compound, Le., a compound which does not
spontaneously
undergo transformation such as by rearrangement, cyclization, elimination,
etc. In certain
embodiments, substituted refers to moieties having substituents replacing two
hydrogen atoms on
the same carbon atom, such as substituting the two hydrogen atoms on a single
carbon. with an
oxo, imino or thioxo group. As used herein, the term "substituted" is
contemplated to include all
permissible substituents of organic compounds. In a broad aspect, the
permissible substituents
include acyclic and cyclic, branched and -unbranched, carbocyclic and
heterocyclic, aromatic and
non-aromatic substituents of organic compounds. The permissible substituents
can be one or
more and the same or different for appropriate organic compounds. For purposes
of this
disclosure, the heteroatoms such as nitrogen may have hydrogen substituents
and/or any
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
permissible substituents of organic compounds described herein which satisfy
the valences of the
heteroatoms.
[0088] In some embodiments, substituents may include any substituents
described herein, for
example: halogen, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2),
imino (=N-H),
oximo (=N-OH), hydrazino (=N-
NH2), -Rb-ORa, -Rb-OC(0)-Ra, -Rb-OC(0)-0Ra, -Rb-OC(0)-N(R1)2, -Rb_N(R1)2,
_Rb_c(0)Ra, _Rb
-C(0)0Ra, -Rb-C(0)N(R1)2, -Rb-0-12c-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-
N(R1)C(0)Ra, -Rb-N
(Ra)S(0)Ra (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)tORa
(where t is 1 or 2),
and -Rb-S(0)tN(R1)2 (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl,
aralkyl, aralkenyl,
aralkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo alkyl,
heterocycloalkylalkyl, heteroaryl, and
heteroarylalkyl any of which may be optionally substituted by alkyl, alkenyl,
alkynyl, halogen,
haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro
(-NO2), imino
(=N-H), oximo (=N-OH), hydrazine (=N-
NH2), -Rb-ORa, -Rb-OC(0)-Ra, -Rb-OC(0)-0Ra, -Rb-OC(0)-N(R1)2, -Rb_N(R1)2,
_Rb_c(0)Ra, _Rb
-C(0)0Ra, -Rb-C(0)N(R1)2, -Rb-0-12c-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-
N(R1)C(0)Ra, -Rb-N
(Ra)S(0)Ra (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)tORa
(where t is 1 or 2)
and -Rb-S(0)tN(R1)2 (where t is 1 or 2); wherein each Ra is independently
selected from
hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each Ra,
valence permitting, may
be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl,
haloalkenyl,
haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H),
oximo (=N-OH),
hydrazine (=N-
NH2), -Rb-ORa, -Rb-OC(0)-Ra, -Rb-OC(0)-0Ra, -Rb-OC(0)-N(R1)2, -Rb_N(R1)2,
_Rb_c(0)Ra, _Rb
-C(0)0Ra, -Rb-C(0)N(R1)2, -Rb-0-12c-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-
N(R1)C(0)Ra, -Rb-N
(Ra)S(0)Ra (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)tORa
(where t is 1 or 2)
and -Rb-S(0)tN(R1)2 (where t is 1 or 2); and wherein each Rb is independently
selected from a
direct bond or a straight or branched alkylene, alkenylene, or alkynylene
chain, and each Rc is a
straight or branched alkylene, alkenylene or alkynylene chain.
[0089] It will be understood by those skilled in the art that substituents can
themselves be
substituted, if appropriate. Unless specifically stated as "unsubstituted,"
references to chemical
moieties herein are understood to include substituted variants. For example,
reference to a
"heteroaryl" group or moiety implicitly includes both substituted and
unsubstituted variants.
[0090] Chemical entities having carbon-carbon double bonds or carbon-nitrogen
double bonds
may exist in Z- or E- form (or cis- or trans- form). Furthermore, some
chemical entities may exist
31
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
in various tautomeric forms. Unless otherwise specified, chemical entities
described herein are
intended to include all Z-, E- and tautomeric forms as well.
[0091] A "tautomer" refers to a molecule wherein a proton shift from one atom
of a molecule to
another atom of the same molecule is possible. The compounds presented herein,
in certain
embodiments, exist as tautomers. In circumstances where tautomerization is
possible, a chemical
equilibrium of the tautomers will exist. The exact ratio of the tautomers
depends on several
factors, including physical state, temperature, solvent, and pH. Some examples
of tautomeric
\IN-\
H H
\
N H2 H
\ NH2 N H
\2.N
csr¨ N H iscs
ssi\J N,
N N HN N' N zs_ ,N H
rrc.-N s 5 N 5 H
I
equilibrium include: H NOH 0
[0092] The compounds disclosed herein, in some embodiments, are used in
different enriched
isotopic forms, e.g., enriched in the content of 2H, 3H, 11,,, 13
C and/or 14C. In one particular
embodiment, the compound is deuterated in at least one position. Such
deuterated forms can be
made by the procedure described in U.S. Patent Nos. 5,846,514 and 6,334,997.
As described in
U.S. Patent Nos. 5,846,514 and 6,334,997, deuteration can improve the
metabolic stability and or
efficacy, thus increasing the duration of action of drugs.
[0093] Unless otherwise stated, structures depicted herein are intended to
include compounds
which differ only in the presence of one or more isotopically enriched atoms.
For example,
compounds having the present structures except for the replacement of a
hydrogen by a
deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched
carbon are within
the scope of the present disclosure.
[0094] The compounds of the present disclosure optionally contain unnatural
proportions of
atomic isotopes at one or more atoms that constitute such compounds. For
example, the
compounds may be labeled with isotopes, such as for example, deuterium (2H),
tritium (3H),
iodine-125 (1251) or carbon-14 (..,) Isotopic substitution with
2H, HC, 13C, 14C, 15C, 12N, 13N,
32
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
15 '6N, 16 17 14 15 16 17 18 33 34 35 36
35 37 79 81 125
N, N, F, F, F, F, F, S, S, S, S, Cl, Cl, Br, Br, I are all
contemplated. All isotopic variations of the compounds of the present
invention, whether
radioactive or not, are encompassed within the scope of the present invention.
[0095] In certain embodiments, the compounds disclosed herein have some or all
of the 1H atoms
replaced with 2H atoms. The methods of synthesis for deuterium-containing
compounds are
known in the art and include, by way of non-limiting example only, the
following synthetic
methods.
[0096] Deuterium substituted compounds are synthesized using various methods
such as
described in: Dean, Dennis C.; Editor. Recent Advances in the Synthesis and
Applications of
Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm.
Des., 2000;
6(10)[ 2000, 110 pp; George W.; Varma, Rajender S. The Synthesis of
Radiolabeled Compounds
via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and
Evans, E. Anthony.
Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.
[0097] Deuterated starting materials are readily available and are subjected
to the synthetic
methods described herein to provide for the synthesis of deuterium-containing
compounds. Large
numbers of deuterium-containing reagents and building blocks are available
commercially from
chemical vendors, such as Aldrich Chemical Co.
[0098] Compounds of the present invention also include crystalline and
amorphous forms of
those compounds, pharmaceutically acceptable salts, and active metabolites of
these compounds
having the same type of activity, including, for example, polymorphs,
pseudopolymorphs,
solvates, hydrates, unsolvated polymorphs (including anhydrates),
conformational polymorphs,
and amorphous forms of the compounds, as well as mixtures thereof.
[0099] The phrases "parenteral administration" and "administered parenterally"
as used herein
means modes of administration other than enteral and topical administration,
usually by injection,
and includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous,
subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and
intrasternal injection and
infusion.
[0100] The phrase "pharmaceutically acceptable" is employed herein to refer to
those
compounds, materials, compositions, and/or dosage forms which are, within the
scope of sound
medical judgment, suitable for use in contact with the tissues of human beings
and animals
without excessive toxicity, irritation, allergic response, or other problem or
complication,
commensurate with a reasonable benefit/risk ratio.
33
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0101] The phrase "pharmaceutically acceptable excipient" or "pharmaceutically
acceptable
carrier" as used herein means a pharmaceutically acceptable material,
composition or vehicle,
such as a liquid or solid filler, diluent, excipient, solvent or encapsulating
material. Each carrier
must be "acceptable" in the sense of being compatible with the other
ingredients of the
formulation and not injurious to the patient. Some examples of materials which
can serve as
pharmaceutically acceptable carriers include: (1) sugars, such as lactose,
glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and its
derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository
waxes; (9) oils, such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (10)
glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and
polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13)
agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid;
(16) pyrogen-
free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer
solutions; and (21) other non-toxic compatible substances employed in
pharmaceutical
formulations.
[0102] An antigen can elicit an immune response. An antigen can be a protein,
polysaccharide,
lipid, or glycolipid, which can be recognized by an immune cell, such as a T
cell or a B cell.
Exposure of immune cells to one or more of these antigens can elicit a rapid
cell division and
differentiation response resulting in the formation of clones of the exposed T
cells and B cells. B
cells can differentiate into plasma cells which in turn can produce antibodies
which selectively
bind to the antigens.
[0103] The terms "cancer" and "tumor" relate to the physiological condition in
mammals
characterized by deregulated cell growth. Cancer is a class of diseases in
which a group of cells
display uncontrolled growth or unwanted growth. Cancer cells can also spread
to other locations,
which can lead to the formation of metastases. Spreading of cancer cells in
the body can, for
example, occur via lymph or blood. Uncontrolled growth, intrusion and
metastasis formation are
also termed malignant properties of cancers. These malignant properties
differentiate cancers
from benign tumors, which typically do not invade or metastasize.
[0104] In cancer, there are four general groups of tumor antigens: (i) viral
tumor antigens which
can be identical for any viral tumor of this type, (ii) carcinogenic tumor
antigens which can be
specific for patients and for the tumors, (iii) isoantigens of the
transplantation type or tumor-
specific transplantation antigens which can be different in all individual
types of tumor but can
be the same in different tumors caused by the same virus; and (iv) embryonic
antigens.
34
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0105] As a result of the discovery of tumor antigens, tumor antigens have
become important in
the development of new cancer treatments that can specifically target the
cancer. This has led to
the development of antibodies directed against these tumor antigens.
[0106] In addition to the development of antibodies against tumor antigens for
cancer treatment,
antibodies that target immune cells to boost the immune response have also
been developed. For
example, an anti-CD40 antibody that is a CD40 agonist can be used to activate
dendritic cells to
enhance the immune response.
[0107] Cluster of Differentiation 40 (CD40) is a member of the Tumor Necrosis
Factor
Receptor (TNF-R) family. CD40 can be a 50 kDa cell surface glycoprotein that
can be
constitutively expressed in normal cells, such as monocytes, macrophages, B
lymphocytes,
dendritic cells, endothelial cells, smooth muscle cells, fibroblasts and
epithelium, and in tumor
cells, including B-cell lymphomas and many types of solid tumors. Expression
of CD40 can be
increased in antigen presenting cells in response to IL-10p, IFN-y, GM-CSF,
and LPS induced
signaling events.
[0108] Humoral and cellular immune responses can be regulated, in part, by
CD40. For
example, in the absence of CD40 activation by its cognate binding partner,
CD40 Ligand
(CD4OL), antigen presentation can result in tolerance. However, CD40
activation can ameliorate
tolerance. In addition, CD40 activation can positively impact immune responses
by enhancing
antigen presentation by antigen presenting cells (APC), increasing cytokine
and chemokine
secretion, stimulating expression of and signaling by co-stimulatory
molecules, and activating the
cytolytic activity of different types of immune cells. Accordingly, the
interaction between CD40
and CD4OL can be essential to maintain proper humoral and cellular immune
responses.
[0109] The intracellular effects of CD40 and CD4OL interaction can include
association of the
CD40 cytoplasmic domain with TRAFs (TNF-R associated factors), which can lead
to the
activation of NFKB and Jun/AP1 pathways. While the response to activation of
NFKB and
Jun/AP1 pathways can be cell type-specific, often such activation can lead to
increased
production and secretion of cytokines, including IL-6, IL-8, IL-12, IL-15;
increased production
and secretion of chemokines, including MIPla and 0 and RANTES; and increased
expression of
cellular adhesion molecules, including ICAM. While the effects of cytokines,
chemokines and
cellular adhesion molecules can be widespread, such effects can include
enhanced survival and
activation of T cells.
[0110] In addition to the enhanced immune responses induced by CD40
activation, CD40
activation can also be involved in chemokine- and cytokine-mediated cellular
migration and
differentiation; activation of immune cells, including monocytes; activation
of and increased
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cytolytic activity of immune cells, including cytolytic T lymphocytes and
natural killer cells;
induction of CD40-positive tumor cell apoptosis and enhanced immunogenicity of
CD40-positive
tumors. In addition, CD40 can initiate and enhance immune responses by many
different
mechanisms, including, inducing antigen-presenting cell maturation and
increased expression of
costimulatory molecules, increasing production of and secretion of cytokines,
and enhancing
effector functions.
[0111] CD40 activation can be effective for inducing immune-mediated antitumor
responses.
For example, CD40 activation reverses host immune tolerance to tumor-specific
antigens which
leads to enhanced antitumor responses by T cells. Such antitumor activity can
also occur in the
absence of immune cells. Similarly, antitumor effects can occur in response to
anti-CD40
antibody-mediated activation of CD40 and can be independent of or can involve
antibody-
dependent cellular cytotoxicity (ADCC). In addition to other CD40-mediated
mechanisms of
antitumor effects, CD4OL-stimulation can cause dendritic cell maturation and
stimulation.
CD4OL-stimulated dendritic cells can contribute to the antitumor response.
Furthermore,
vaccination strategies including CD40 can result in regression of CD40-
positive and CD40-
negative tumors.
[0112] CD40 activating antibodies (e.g., anti-CD40 activating monoclonal
antibodies) can be
useful for treatment of tumors. This can occur through one or more mechanisms,
including cell
activation, antigen presentation, production of cytokines and chemokines,
amongst others. For
example, CD40 antibodies activate dendritic cells, leading to processing and
presentation of
tumor antigens as well as enhanced immunogenicity of CD40-positive tumor
cells. Not only can
enhanced immunogenicity result in activation of CD40-positive tumor specific
CD4+ and CD8+ T
cells, but further antitumor activity can include, recruitment and activation
monocytes, enhanced
cytolytic activity of cytotoxic lymphocytes and natural killer cells as well
as induction of
apoptosis or by stimulation of a humoral response so as to directly target
tumor cells. In addition,
tumor cell debris, including tumor-specific antigens, can be presented to
other cells of the
immune system by CD40-activated antigen presenting cells.
[0113] Since CD40 can be important in an immune response, there is a need for
enhanced CD40
meditated signaling events to provide reliable and rapid treatment options to
patients suffering
from diseases which may be ameliorated by treatment with CD40-targeted
therapeutic strategies.
[0114] The CD40 mediated immune response can be further enhanced by targeting
CD40
activation to the localized tumor site(s) through pairing with a tumor antigen
binding domain.
Such targeted CD40 activation and recruitment of immune cells to tumor cells
may provide the
advantage of maintaining therapeutic effectiveness with a lower dosage of a
CD40 activating
36
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
antibody construct or conjugate. A lower dosage may help mitigate any side
effects of systemic
CD40 activation such as cytokine release syndrome, which has been observed in
some subjects
treated with the agonistic CD40 monoclonal antibodies such as CP-870,893,
dacetuzumab, Chi
Lob 7/4, SEA-CD40, ADC-1013, 3C3, or 3G5. Systemic CD40 activation may also
pose a risk of
autoimmunity by causing APCs to break tolerance of autoantigens. For example,
autoreactive T
cells that manage to evade thymic selection may persist in the periphery in a
state of tolerance
against autoantigens, but CD40 activation can cause them to break tolerance
and exhibit an
autoimmune response. Accordingly, there is an important need for therapeutic,
clinically relevant
targeted CD40 activation that is enhanced at the localized tumor site relative
to systemic
activation. The presently described conjugate can be utilized as a safe and
effective strategy to
enhance the immune response. A conjugate can comprise an antigen binding
domain and a CD40
binding domain, wherein the antigen binding domain specifically binds to a
tumor antigen,
wherein the CD40 binding domain comprises a CD40 agonist. This combination of
a tumor
antigen binding domain and a CD40 agonist can provide enhanced CD40 activation
and
recruitment of immune cells to the localized tumor site.
[0115] In addition to targeting immune cells to boost the immune response
using anti-CD40
antibodies, antibodies that target other antigens expressed by immune cells
have been developed.
For example, an anti-DEC205 antibody, an anti-CD36 mannose scavenger receptor
1 antibody,
an anti-CLEC9A antibody, CLEC12A, an anti-DC-SIGN antibody, an anti-BDCA-2
antibody, an
anti-OX4OL antibody, an anti-41BBL antibody, an anti-CD204 antibody, an anti-
MARCO
antibody, an anti-CLEC5A antibody, an anti-Dectin 1 antibody, and anti-Dectin
2 antibody, an
anti-CLEC10A antibody, an anti-CD206 antibody, an anti-CD64 antibody, an anti-
CD32A
antibody, an anti-CD16A antibody, an anti-HVEM antibody, an anti-PD-Li
antibody, an anti-
CD32B antibody or an anti-CD47 antibody can be used to target, respectively,
surface DEC-205,
CD36 mannose scavenger receptor 1, CLEC9A, CLEC12A, DC-SIGN, BDCA-2, OX4OL,
41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A,
CD16A, HVEM, PD-L1, or CD32B molecules expressed by antigen presenting cells
or CD47
molecules expressed by T cells.
[0116] Cluster of Differentiation 205 (CD205 or DEC-205) is a member of the C-
type
multilectin family of endocytic receptors, which can include the macrophage
mannose receptor
(MMR) and the phospholipase A2 receptor (PLA2R). DEC-205 can be a 205 kDa
endocytic
receptor highly expressed in cortical thymic epithelial cells, thymic
medullary dendritic cells
(CD11c+ CD8+), subpopulations of peripheral dendritic cells (CD11c+ CD8+). The
DEC-205+
CD11c+ CD8+ dendritic cells (DCs) can function in cross-presentation of
antigens derived from
37
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
apoptotic cells. Additionally, DEC-205 can be significantly upregulated during
DC maturation.
DEC-205 can also be expressed at moderate levels in B cells and low levels in
macrophages and
T cells.
[0117] After antigen binding to DEC-205, the receptor-antigen complex can be
internalized
whereupon the antigen can be processed and be presented on the DC surface by a
major
histocompatibility complex class II (MHC II) or MHC class I. DEC-205 can
deliver antigen to
DCs for antigen presentation on MHC class II and cross-presentation on MHC
class I. DEC-205
mediated antigen delivery for antigen presentation in DCs without an
inflammatory stimulus can
result in tolerance. Conversely, DEC-205 mediated antigen delivery in DCs in
the presence of a
maturational stimulus (e.g. a CD40 agonist) can result in long-term immunity
via activation of
antigen-specific CD4+ and CD8+ T cells.
[0118] CD36 mannose scavenger receptor 1 is an oxidized LDL receptor with two
transmembrane domains located in the caveolae of the plasma membrane. It can
be classified as a
Class B scavenger receptor, which can be characterized by involvement in the
removal of foreign
substances and waste materials. This receptor can also be involved in cell
adhesion, phagocytosis
of apoptotic cells, and metabolism of long-chain fatty acids.
[0119] CLEC9A is a group V C-type lectin receptor. This receptor can be
expressed as on
myeloid lineage cells, and can be characterized as an activation receptor.
[0120] CLEC12A is a member of the C-type lectin/C-type lectin like domain
super family that
can be a negative regulator of granulocyte and monocyte function. It can also
be involved in cell
adhesion, cell-cell signaling, and glycoprotein turnover, and can play a role
in the inflammatory
response.
[0121] Dendritic cell-specific inter cellular adhesion molecule-3-grabbing non-
integrin (DC-
SIGN) or CD209, is a C-type lectin receptor that can be expressed on the
surface of macrophages
and dendritic cells. This receptor can recognize and bind to mannose type
carbohydrates and be
involved in activating phagocytosis, can mediate dendritic cell rolling, and
can be involved in
CD4+ T cell activation.
[0122] BDCA-2 is a C-type lectin that is a membrane protein of plasmacytoid
dendritic cells. It
can be involved in plasmacytoid dendritic cell function, such as ligand
internalization and
presentation.
[0123] OX4OL, which can also be referred to as CD252, is the ligand for CD134
that can be
expressed on dendritic cells. It can be involved in T cell activation.
38
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0124] 41BBL, which can also be referred to as CD137L, is a member of the TNF
superfamily,
and can be expressed on B cells, dendritic cells, activated T cells, and
macrophages. It can
provide co-stimulatory signal for T cell activation and expansion.
[0125] CD204, which can also be referred to as macrophage scavenger receptor
1, is a
macrophage scavenger receptor receptor. The gene for CD204 can encode three
different class A
macrophage scavenger receptor isoforms. The type 1 and type 2 isoforms can be
involved in
binding, internalizing, and processing negatively charged macromolecules, such
as low density
lipoproteins. The type 3 isoform can undergo altered intracellular processing
in which it can be
retained within the endoplasmic reticulum, and has been shown to have a
dominant negative
effect on the type 1 and type 2 isoforms.
[0126] Macrophage receptor with collagenous structure (MARCO), which can also
be referred to
as SCARA2, is a class A scavenger receptor with collagen-like and cysteine-
rich domains. It can
be expressed in macrophages, and can bind to modified low density
lipoproteins. It can be
involved in the removal of foreign substances and waste materials.
[0127] C-type lectin domain family 5 member A (CLEC5A) is a C-type lectin. It
can be involved
in the myeloid lineage activating pathway.
[0128] Dendritic cell-associated c-type lectin-1 (Dectin 1), which can also be
referred to as
CLEC7A, is member of the C-type lectin/C-type lectin-like super family. It can
be expressed by
myeloid dendritic cells, monocytes, macrophages, and B cells, and can be
involved in antifungal
immunity.
[0129] Dendritic cell-associated c-type lectin-2 (Dectin 2), which can also be
referred to as
CLEC6A, is member of the C-type lectin/C-type lectin-like super family. It can
be expressed by
dendritic cells, macrophages, monocytes and neutrophils. It can be involved in
antifungal
immunity.
[0130] CLEC10A, which can also be referred to as CD301, is member of the C-
type lectin/C-
type lectin-like super family. It can be expressed by dendritic cells,
monocytes, and CD33+
myeloid cells, and can be involved in macrophage adhesion and migration.
[0131] CD206, which can also be referred to as macrophage mannose receptor, is
a C-type lectin
type I membrane glycoprotein. It can be expressed on dendritic cells,
macrophages and
endothelial cells, and can act as a pattern recognition receptor and bind high-
mannose structures
of viruses, bacteria, and fungi.
[0132] CD64, which can also be referred to as FcyRI, is a high affinity Fc
receptor for IgG. It can
be expressed by monocytes and macrophages. It can be involved in mediating
phagocytosis,
antigen capture, and antibody dependent cell-mediated cytoxicity.
39
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0133] CD32A, which can also be referred to as FcyRIIa, is a low affinity Fc
receptor. It can be
expressed on monocytes, granulocytes, B cells, and eosinophils. It can be
involved in
phagocytosis, antigen capture, and antibody dependent cell-mediated
cytoxicity.
[0134] CD16A, which can also be referred to as FcyRIIIa, is low affinity Fc
receptor. It can be
expressed on NK cells, and can be involved in phagocytosis and antibody
dependent cell-
mediated cytotoxicity.
[0135] Herpesvirus entry mediator (HVEM), which can also be referred to as
CD270, is a
member of the TNF-receptor superfamily. It can be expressed on B cells,
dendritic cells, T cells,
NK cells, CD33+ myeloid cells, and monocytes. It can be involved in activating
the immune
response.
[0136] CD32B, which can also be referred to as FcyRIIb, is a low affinity Fc
receptor. It can be
expressed on B cells and myeloid dendritic cells. It can be involved in
inhibiting maturation and
cell activation of dendritic cells.
[0137] The HER2/neu (human epidermal growth factor receptor 2/receptor
tyrosine-protein
kinase erbB-2) is part of the human epidermal growth factor family.
Overexpression of this
protein can be shown to play an important role in the progression of cancer,
for example, breast
cancer. The HER2/neu protein can function as a receptor tyrosine kinase and
autophosphorylates
upon dimerization with binding partners. HER2/neu can activate several
signaling pathways
including, for example, mitogen-activated protein kinase, phosphoinositide 3-
kinase,
phospholipase Cy, protein kinase C, and signal transducer and activator of
transcription (STAT).
Examples of antibodies that can target and inhibit HER2/neu can include
trastuzumab and
pertuzumab.
[0138] EGFR (epidermal growth factor receptor) encodes a member of the human
epidermal
growth factor family. Mutations that can lead to EGFR overexpression or over
activity can be
associated with a number of cancers, including squamous cell carcinoma and
glioblastomas.
EGFR can function as a receptor tyrosine kinase and ligand binding can trigger
dimerization with
binding partners and autophosphorylation. The phosphorylated EGFR can then
activate several
downstream signaling pathways including mitogen-activated protein kinase,
phosphoinositide 3-
kinase, phospholipase Cy, protein kinase C, and signal transducer and
activator of transcription
(STAT). Examples of antibodies that can target and inhibit EGFR can include
cetuximab,
panutumumab, nimotuzumab, and zalutumumab. One mutant variant of EGFR is
EGFRvIII
(epidermal growth factor receptor variant III). EGFRvIII can be the result of
an EGFR gene
rearrangement in which exons 2-7 of the extracellular domain are deleted. This
mutation can
result in a mutant receptor incapable of binding to any known ligand. The
resulting receptor can
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
engage in a constitutive low-level signaling and can be implicated in tumor
progression.
Examples of antibodies that can target EGFRvIII can include AMG595 and ABT806.
[0139] C-Met (hepatocyte growth factor receptor) encodes a member of the
receptor tyrosine
kinase family of proteins. C-Met overexpression and over activity can be
implicated in various
cancers including lung adenocarcinomas, and high c-Met levels can be
associated with poor
patient outcome. Binding of hepatocyte growth factor can induce dimerization
and
autophosphorylation of c-Met. The c-Met receptor can activate various
downstream signaling
pathways including mitogen-activated protein kinase, phosphoinositide 3-
kinase, and protein
kinase C pathways. The antibody onartuzumab can target and inhibit c-Met.
[0140] HER3 (human epidermal growth factor receptor 3) encodes a member of the
human
epidermal growth factor receptor family. Ligand binding can induce
dimerization and
autophosphorylation of cytoplasmic tyrosine residues that then can recruit
signaling proteins for
downstream signaling pathway activation including mitogen-activated protein
kinase and
phosphoinoside 3-kinase pathways. HER3 can play an active role in cell
proliferation and
survival, and can be overexpressed, overactive, and/or mutated in various
cancers. For example,
HER3 can be overexpressed in breast, ovarian, prostate, colon, pancreas,
stomach, oral, and lung
cancers. The antibody patritumab can target and inhibit HER3.
[0141] MUC1 (mucin 1, cell surface associated) encodes a member of the mucin
family of
glycosylated proteins that can play an important role in cell adhesion and
forming protective
mucosal layers on epithelial surfaces. MUC1 can be proteolytically cleaved
into alpha and beta
subunits that form a heterodimeric complex with the N-terminal alpha subunit
providing cell-
adhesion functionality and the C-terminal beta subunit modulating cell
signaling pathways
including the mitogen activated map kinase pathway. MUC1 can play a role in
cancer
progression, for example, by regulating TP53-mediated transcription. MUC1
overexpression,
aberrant intracellular localization, and glycosylation changes can all be
associated with
carcinomas including pancreatic cancer cells. The antibody clivatuzumab can
target MUC1.
[0142] MUC16 (mucin 16, cell surface associated) encodes the largest member of
the mucin
family of glycosylated proteins that can play an important role in cell
adhesion and forming
protective mucosal layers on epithelial surfaces. MUC16 can be a highly
glycosylated 2.5MDa
transmembrane protein that can provide a hydrophilic lubricating barrier on
epithelial cells. The
cytoplasmic tail of MUC16 can be involved with various signaling pathways
including the
JAK2-STAT3 and Src kinase pathways. A peptide epitope of MUC16 can be used as
biomarker
for detecting ovarian cancer. Elevated expression of MUC16 can be present in
advanced ovarian
cancers and pancreatic cancers. The antibody sofituzumab can target MUC16.
41
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0143] EPCAM (epithelial cell adhesion molecule) encodes a transmembrane
glycoprotein that
can be frequently and highly expressed in carcinomas and tumor-initiating
cells. EPCAM can
also be a pluripotent stem cell marker. EPCAM can modulate a variety of
pathways including
cell-cell adhesion, cellular proliferation, migration, invasion, maintenance
of a pluripotent state,
and differentiation in the context of tumor cells. The antibodies edrecolomab
and adecatumumab
can target EPCAM.
[0144] MSLN (mesothelin) encodes a 40 kDa cell GPI-anchored membrane surface
protein
believed to function in cell adhesion. MSLN is overexpressed in mesothelioma
and certain types
of pancreatic, lung, and ovarian cancers. MSLN-related peptides that circulate
in serum of
patients suffering from pleural mesothelioma are used as biomarkers for
monitoring the disease.
MSLN may promote metastasis by inducing matrix metalloproteinase 7 and 9
expression. The
monoclonal antibody anetumab has been developed to target MSLN.
[0145] CA6 (carbonic anhydrase VI) encodes one of several isozymes of carbonic
anhydrase.
CA6 is found in salivary glands and may play a role in the reversible
hydration of carbon dioxide.
CA6 is expressed in human serous ovarian adenocarcinomas. The monoclonal
antibody huDS6
has been developed to target CA6.
[0146] NAPI2B (sodium/phosphate cotransporter 2B) encodes a type II sodium-
phosphate
cotransporter. NAPI2B is highly expressed on the tumor surface in lung,
ovarian, and thyroid
cancers as well as in normal lung pneumocytes. The monoclonal antibody
lifastuzumab has been
developed to target NAPI2B.
[0147] TROP2 (trophoblast antigen 2) encodes a transmembrane glycoprotein that
acts as an
intracellular calcium signal transducer. TROP2 binds to multiple factors such
as IGF-1, claudin-1,
claudin-7, cyclin D1, and PKC. TROP2 including intracellular calcium signaling
and the mitogen
activated protein kinase pathway. TROP 2 plays a role in cell self-renewal,
proliferation, invasion,
and survival. Discovered first in trophoblast cells that have the ability to
invade uterine decidua
during placental implantation, TROP2 overexpression has been shown to be
capable of
stimulating cancer growth. TROP2 overexpression has been observed in breast,
cervix, colorectal,
esophagus, lung, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma, Raji
Burkitt
lymphoma, oral squamous cell, ovarian, pancreatic, prostate, stomach, thyroid,
urinary bladder,
and uterine carcinomas. The monoclonal antibody sactuzumab has been developed
to target
TROP2.
[0148] CEA (carcinoembryonic antigen) encodes a family of related
glycoproteins involved in
cell adhesion. CEA is a biomarker for gastrointestinal cancers and may promote
tumor
development by means of its cell adhesion function. CEA levels have been found
to be elevated
42
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
in serum of individuals with colorectal carcinoma. CEA levels have also been
found to be
elevated in gastric carcinoma, pancreatic carcinoma, lung carcinoma, breast
carcinoma, and
medullary thyroid carcinoma. The monoclonal antibodies PR1A3 and Ab2-3 have
been
developed to target CEA.
[0149] CLDN18.2 (claudin 18) encodes a member of the claudin family of
integral membrane
proteins. CLDN18.2 is a component of tight junctions that create a physical
barrier to prevent
diffusion of solutes and water through the paracellular space between
epithelial cells. CLDN18.2
is overexpressed in infiltrating ductal adenocarcinomas, but is reduced in
some gastric
carcinomas. The monoclonal antibody claudiximab has been developed to target
CLDN18.2.
[0150] FAP (fibroblast activation protein, alpha) encodes a homodimeric
integral membrane
protein from a family of serine proteases. FAP is believed to play a role in
many processes
including tissue remodeling, fibrosis, wound healing, inflammation, and tumor
growth. FAP
enhances tumor growth and invasion by promoting angiogenesis, collagen fiber
degradation and
apoptosis, and by downregulating the immune response. FAP is selectively
expressed on
fibroblasts within the tumor stroma. The monoclonal antibody sibrotuzumab has
been developed
to target FAP.
[0151] EphA2 (EPH Receptor A2) encodes a member of the ephrin receptor
subfamily of the
protein-tyrosine kinase family. EphA2 binds to ephrin-A ligands. Activation of
EphA2 receptor
upon ligand binding can result in modulation of migration, integrin-mediated
adhesion,
proliferation, and differentiation. EphA2 is overexpressed in various cancers
including breast,
prostate, urinary bladder, skin, lung, ovarian, and brain cancers. High EphA2
expression is also
correlated with poor prognosis. The monoclonal antibodies DS-8895a optl, DS-
8895 opt2, and
Anti-EphA2 of MEDI-547 have been developed to target EphA2.
[0152] RON (macrophage stimulating 1 receptor) encodes a cell surface receptor
for macrophage
stimulating protein (MSP) with tyrosine kinase activity and belongs to the MET
proto-oncogene
family. RON has significant structural similarity and sequence homology with
the cancer-related
gene C-MET. RON plays a significant role in KRAS oncogene addiction and has
also been
shown to be overexpressed in pancreatic cancers. Altered Ron expression and
activation has been
associated with decreased survival and cancer progression in various cancers
including gastric,
colon, breast, bladder, renal cell, ovarian, and hepatocellular cancers. The
monoclonal antibody
narnatumab has been developed to target RON.
[0153] LY6E (lymphocyte antigen 6 complex, locus E) encodes an interferon
alpha-inducible
GPI-anchored cell membrane protein. LY6E is overexpressed in numerous cancers
including
43
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
lung, gastric, ovarian, breast, kidney, pancreatic, and head and neck
carcinomas. The monoclonal
antibody RG7841 has been developed to target LY6E.
[0154] FRA (folate receptor alpha) encodes a GPI-anchored cell surface
glycoprotein. FRA binds
folic acid, a molecule needed for cell growth and DNA synthesis, and mediates
its internalization
via receptor-mediated endocytosis. FRA is overexpressed in various cancers
including prostate,
breast, ovarian, pancreatic, mesothelioma, non-small cell lung carcinoma, and
head and neck
cancer. FRA expression has also been found to enhance tumor cell
proliferation. The monoclonal
antibodies farletuzumab and mirvetuximab have been developed to target FRA.
[0155] PSMA (prostate specific membrane antigen) is a type II transmembrane
glycoprotein
belonging to the M28 peptidase family that is expressed in all types of
prostate tissues. PSMA is
upregulated in cancer cells within the prostate and is used as a marker for
prostate cancer. PSMA
expression may also serve as a predictor of disease recurrence in prostate
cancer patients. The
monoclonal antibodies J591 variant 1 and J591 variant 2 have been developed to
target PSMA.
[0156] DLL3 (delta-like 3) encodes a ligand in the Notch signaling pathway
that is associated
with neuroendocrine cancer. DLL3 is most highly expressed in the fetal brain
and is involved in
somitogenesis in the paraxial mesoderm. DLL3 is expressed on tumor cell
surfaces but not in
normal tissues. The monoclonal antibody rovalpituzumab has been developed to
target DLL3.
[0157] PTK7 (tyrosine protein kinase-like 7) encodes a receptor tyrosine
kinase that lacks
catalytic tyrosine kinase activity but is nevertheless capable of signal
transduction. PTK7
interacts with the WNT signaling pathway, which itself has important roles in
epithelial
mesenchymal transition and various cancers such as breast cancer. PTK7
overexpression has
been associated with patient prognosis depending on the cancer type. The
monoclonal antibodies
PF-06647020 and the anti-PTK7 antibody described by SEQ ID NO 440 and 445 have
been
developed to target PTK7.
[0158] LIV1 (LIV-1 protein, estrogen regulated) encodes a member of the LIV-1
subfamily of
ZIP (Zrt-, Irt-like proteins) zinc transporters. LIV1 is an estrogen regulated
protein that transports
zinc and/or other ions across the cell membrane. Elevated levels of LIV1 have
been shown in
estrogen receptor positive breast cancers, and LIV1 is used as a marker of ER-
positive cancers.
LIV1 has also been implicated as a downstream target of the STAT3
transcription factor and as
playing an essential role in the nuclear localization of the Snail
transcription factor that
modulates epithelial-to-mesenchymal transition. The monoclonal antibody
Ladiratuzumab has
been developed to target LIV1.
[0159] ROR1 (receptor tyrosine kinase-like orphan receptor 1) encodes a member
of the ROR
family of orphan receptors. ROR1 has been found to bind Wnt5a, a non-canonical
Wnt via a
44
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Frizzled domain (FZD), and plays an important role in skeletal,
cardiorespiratory, and
neurological development. ROR1 expression is predominantly restricted to
embryonic
development and is absent in most mature tissues. In contrast, ROR1 expression
is upregulated in
B-Cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin
lymphoma,
and myeloid malignancies. The monoclonal antibody cirmtuzumab has been
developed to target
ROR1.
[0160] MAGE-A3 (melanoma-associated antigen 3) encodes a member of the
melanoma-
associated antigen gene family. The function of MAGE-A3 is not known, but its
elevated
expression has been observed in various cancers including melanoma, non-small
cell lung cancer,
and in putative cancer stem cell populations in bladder cancer. The monoclonal
antibody
described by SEQ ID NO 479 and 484 has been developed to target MAGE-A3.
[0161] NY-ESO-1 (New York esophageal squamous cell carcinoma 1) encodes a
member of the
cancer-testis family of proteins. Cancer-testis antigen expression is normally
restricted to
testicular germ cells in adult tissues, but has been found to be aberrantly
expressed in various
tumors including soft tissue sarcomas, melanoma, epithelial cancers, and
myxoid and round cell
liposarcomas. The monoclonal antibody described by SEQ ID NO 492 and 497 has
been
developed to target NY-ESO-1.
[0162] Immune-stimulatory molecular motifs, such as Pathogen-Associated
Molecular Pattern
molecules (PAMPs), can be recognized by receptors of the innate immune system,
such as Toll-
like receptors (TLRs), Nod-like receptors, C-type lectins, and RIG-I-like
receptors. These
receptors can be transmembrane and intra-endosomal proteins which can prime
activation of the
immune system in response to infectious agents such as pathogens. Similar to
other protein
families, there are many different TLRs, including TLR4, TLR7 and TLR8.
Several agonists
targeting activation of different TLRs have been tried in various
immunotherapies, including
vaccine adjuvants and in cancer immunotherapies. However, therapeutic use of
PAMPs and
DAMPs or other mechanisms of intervention can be limited because systemic
activation of
PAMP and DAMP signaling pathways can have life-threatening consequences due to
cytokine
syndrome-induced or cytokine storm-induced toxic shock syndrome. Accordingly,
there is a
critical need for therapeutic, clinically relevant targeted delivery of PAMP
and DAMP agonists
for safe and effective strategies to enhance immune responses.
[0163] The presently described antibody constructs and conjugates can be
utilized as strategy to
enhance immune responses. A conjugate can comprise an antibody construct and
an immune-
stimulatory compound. A conjugate can comprise a first binding domain, a
second binding
domain, and an immune-stimulatory compound. A conjugate can comprise a first
binding domain,
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
a second binding domain, an Fc domain, and an immune-stimulatory compound. An
antibody
construct can comprise a first binding domain, a second binding domain, and a
third binding
domain. An antibody construct can comprise a first binding domain, a second
binding domain, a
third binding domain, and an Fc domain, wherein the first binding domain is
attached to the Fc
domain, wherein the second binding domain is attached to the Fc domain, and
wherein the third
binding domain is attached to a C-terminal end of a light chain of the first
binding domain. A
conjugate can comprise a first binding domain, a second binding domain, a
third binding domain,
and an immune-stimulatory compound. A conjugate can comprise a first binding
domain, a
second binding domain, a third binding domain, an Fc domain, and an immune-
stimulatory
compound, wherein the first binding domain is attached to the Fc domain,
wherein the second
binding domain is attached to the Fc domain, and wherein the third binding
domain is attached to
a C-terminal end of a light chain of the first binding domain. A conjugate can
comprise a first
binding domain, a second binding domain, a third binding domain, an Fc domain,
and an
immune-stimulatory compound, wherein the first binding domain is attached to
the Fc domain,
wherein the second binding domain is attached to the Fc domain, and wherein
the third binding
domain is attached to a C-terminal end of a light chain of the first binding
domain.
Binding Domains
[0164] A conjugate or antibody construct can contain one or more binding
domains. A conjugate
or antibody construct can comprise a first binding domain. A conjugate or
antibody construct can
comprise a second binding domain. A binding domain can specifically bind to a
molecule on a
cell surface or a fragment thereof. A binding domain can specifically bind to
an antigen on a cell
surface, for example, of a tumor cell, of an antigen presenting cell such as a
dendritic cell or
macrophage or other immune cell cell such as a T cell. In some embodiments, an
immune cell is
a T cell, B cell, NK cell, or NKT cell. In some embodiments, an immune cell is
an antigen
presenting cell. In some embodiments, an immune cell is not an antigen
presenting cell. A
binding domain can specifically bind to a molecule, wherein the molecule
comprises an antigen.
A binding domain can be a cell surface receptor agonist. A binding domain can
be an antigen
binding domain. An antigen binding domain can be a cell surface receptor
agonist. An antigen
binding domain can be a domain that can specifically bind to an antigen. An
antigen binding
domain can specifically bind to a tumor antigen. An antigen binding domain can
be an antigen-
binding portion of an antibody or an antibody fragment. An antigen binding
domain can be one
or more fragments of an antibody that can retain the ability to specifically
bind to an antigen. An
antigen binding domain can be any antigen binding fragment. An antigen binding
domain can
recognize a single antigen. A conjugate can comprise, for example, two, three,
four, five, six,
46
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
seven, eight, nine, ten, or more antigen binding domains. A conjugate or
antibody construct can
comprise two antigen binding domains in which each antigen binding domain can
recognize the
same antigen. A conjugate or antibody construct can comprise two antigen
binding domains in
which each antigen binding domain can recognize different antigens. A
conjugate or antibody
construct can comprise three antigen binding domains in which each antigen
binding domain can
recognize different antigens. A conjugate or antibody construct can comprise
three antigen
binding domains in which two of the antigen binding domains can recognize the
same antigen.
An antigen binding domain can be in a scaffold, in which a scaffold is a
supporting framework
for the antigen binding domain. An antigen binding domain can be in a non-
antibody scaffold.
An antigen binding domain can be in an antibody scaffold or antibody-like
scaffold. A conjugate
or antibody construct can comprise an antigen binding domain in a scaffold.
The conjugate or
antibody construct can comprise an Fc fusion protein product (also referred to
as a fusion
peptide). In some embodiments, the antibody construct is a fusion peptide or
the antibody
construct of a conjugate is a fusion peptide. For example, an antigen binding
domain and an Fc
domain can be expressed as fusion peptide. Two antigen binding domains and an
Fc domain can
be expressed as a fusion peptide.
[0165] The conjugates or antibody constructs described herein can comprise a
binding domain
that can specifically bind to a tumor antigen. A tumor antigen can be a tumor
specific antigen
and/or a tumor associated antigen. As described herein, a "tumor antigen" can
refer to a
molecular marker that can be expressed by a neoplastic tumor cell and/or
within a tumor
microenvironment. The molecular marker can be a cell surface receptor. For
example, a tumor
associated antigen can be an antigen expressed on a cell associated with a
tumor, such as a
neoplastic cell, stromal cell, endothelial cell, fibroblast, or tumor-
infiltrating immune cell. For
example, the tumor associated antigen Her2/Neu can be overexpressed by certain
types of breast
and ovarian cancer. A tumor antigen can also be ectopically expressed by a
tumor and contribute
to deregulation of the cell cycle, reduced apoptosis, metastasis, or escape
from immune
surveillance. Tumor associated antigens can generally be proteins or
polypeptides derived
therefrom, but can be glycans, lipids, or other small organic molecules.
Additionally, a tumor
antigen can arise through increases or decreases in post-translational
processing exhibited by a
cancer cell compared to a normal cell, for example, protein glycosylation,
protein lipidation,
protein phosphorylation, or protein acetylation.
[0166] In certain embodiments, a binding domain specifically can bind to a
tumor associated
antigen selected from the following: CD5, CD19, CD20, CD25, CD37, CD30, CD33,
CD45,
CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLA-DR, carcinoembryonic
47
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
antigen (CEA), TAG-72, MUC1, MUC15, MUC16, folate-binding protein, A33, G250,
prostate-
specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-
9,
epidermal growth factor, HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), EGFR,
fibroblast
activation protein (FAP), tenascin, a metalloproteinase, endosialin, vascular
endothelial growth
factor, avf33, WT1, LMP2, HPV E6, HPV E7, Her-2/neu, p53 nonmutant, NY-ES0-1,
GLP-3,
MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase,
survivin, PSA,
hTERT, a Sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP,
AFP, ERG,
NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC,
TRP-2,
fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, MAGE-A3, sLe(animal), CYP1B1,
PLAV1, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic
anhydrase
IX, PAX5, 0Y-TES1, Sperm protein 17, LCK, MAGE C2, MAGE A4, GAGE, TRAILl,
HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4, VEGFR2, MAD-CT-1,
PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EPCAM, CA6, NAPI2B, TROP2, Claudin-6
(CLDN6), Claudin-16 (CLDN16), CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, Uroplakin-
1B
(UPK1B), LIV1, ROR1, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, Fos-related
antigen 1, VEGFR1, endoglin, PD-L1, VTCN1 (B7-H4), VISTA, or a fragment
thereof. In
certain embodiments, a binding domain specifically binds to a tumor associated
antigen
comprising Her2/Neu (CD340).
[0167] In certain embodiments, a binding domain specifically can bind to a
tumor associated
antigen comprising GD2, GD3, GM2, Leg, polysialic acid, fucosyl GM1, GM3, Tn,
STn,
sLe(animal), or GloboH. In certain embodiments, a binding domain specifically
can bind to a
tumor associated antigen comprising at least 80%, 90%, 95%, 97%, 98%, 99% or
100%
homology to the amino acid sequence of CD5, CD19, CD20, CD25, CD37, CD30,
CD33, CD45,
CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLA-DR, carcinoembryonic
antigen (CEA), TAG-72, MUC1, MUC15, MUC16, folate-binding protein, A33, G250,
prostate-
specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, epidermal growth
factor, HER2,
IL-2 receptor, EGFRvIII (de2-7 EGFR), EGFR, fibroblast activation protein
(FAP), tenascin, a
metalloproteinase, endosialin, vascular endothelial growth factor, avf33, WT1,
LMP2, HPV E6,
HPV E7, Her-2/neu, p53 nonmutant, NY-ESO-1, GLP-3, MelanA/MART1, Ras mutant,
gp100,
p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a Sarcoma
translocation breakpoint
fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen
receptor,
cyclin Bl, MYCN, RhoC, TRP-2, mesothelin (MSLN), PSCA, MAGE Al, MAGE-A3,
CYP1B1,
PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, Carbonic anhydrase IX, PAX5, OY-
TESL Sperm protein 17, LCK, MAGE C2, MAGE A4, GAGE, TRAILl, HMWMAA, AKAP-4,
48
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
SSX2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4,VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-
2, ROR2, CMET, HER3, EPCAM, CA6, NAPI2B, TROP2, Claudin-6 (CLDN6), Claudin-16
(CLDN16), CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, Uroplakin-1B (UPK1B), LIV1,
ROR1,
STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, Fos-related antigen 1, VEGFR1,
endoglin, PD-L1, VTCN1 (B7-H4), VISTA, or a fragment thereof. In certain
embodiments, a
binding domain specifically binds to a tumor associated antigen comprising
Her2/Neu (CD340).
TABLE 1 below shows some exemplary amino acid sequences of these tumor
antigens.
TABLE 1. Exemplary Tumor Antigen Amino Acid Sequences
Antigen SEQ ID Antigen SEQ ID Antigen
SEQ ID
NO: NO: NO:
HIER2 (isoform 1) 850 EPCAM 886 PSMA
(isoform 916
HIER2 (isoform 2) 851 MSLN (isoform -- 887 -- PMSA-4)
HIER2 (isoform 3) 852 Q13421-1) PSMA
(isoform -- 917
HIER2 (isoform 4) 853 MSLN (isoform 2) 888
PMSA')
HIER2 (isoform 5) 854 MSLN (isoform 3) 889 --
PSMA (isoform -- 918
HIER2 (isoform 6) 855 MSLN (isoform 4) 890
PSMA-7)
EGFR (isoform 1) 856 CA6 (isoform 1) 891
PSMA (isoform 919
EGFR (isoform 2) 857 CA6 (isoform 2) -- 892 -- PSMA-8)
EGFR (isoform 3) 858 CA6 (isoform 3) 893
PSMA (isoform 920
EGFR (isoform 4) 859 NAPI2B (isoform 894 PSMA-9)
CMET (isoform 1) 860 1) PSMA (isoform 10) 921
CMET (isoform 2) 861 NAPI2B (isoform 895 DLL3 (isoform 1) -- 922
CMET (isoform 3) 862 2) DLL3 (isoform 2) 923
HIER3 (isoform 1) 863 TROP2 896 PTK7
(isoform 1) 924
HIER3 (isoform 2) 864 CEA (CEACAM5, 897
PTK7 (isoform 2) 925
HIER3 (isoform 3) 865 isoform 1) PTK7
(isoform 3) 926
HIER3 (isoform 4) 866 CEA (CEACAM5, 898 --
PTK7 (isoform 4) -- 927
HIER3 (isoform 5) 867 isoform 2) PTK7
(isoform 5) 928
MUC1 (isoform 1) 868 CLDN18.2 899 PTK7 (isoform 6) 929
MUC1 (Isoform 2) 869 (isoform A2) LIV1 (isoform 1) 930
MUC1 (Isoform 3) 870 EGFRvIII 900 LIV1 (isoform 2) -- 931
MUC1 (Isoform 4) 871 FAP (isoform 1) 901 ROR1 (isoform 1) 932
MUC1 (Isoform 5) 872 FAP (isoform 2) 902 ROR1 (isoform 933
MUC1 (Isoform 6) 873 EphA2 (isoform 1) 903 short)
MUC1 (Isoform Y) 874 EphA2 (isoform 2) 904 ROR1 (isoform 3) 934
MUC1 (Isoform 8) 875 RON (isoform 905 MAGE-A3 935
MUC1 (Isoform 9) 876 RON) NY-ESO-1 936
MUC1 (Isoform F) 877 RON (isoform 906 LRRC15 (isoform 937
MUC1 (Isoform Y- 878 delta) 1)
LSP) RON (isoform 907 LRRC15 (isoform 938
MUC1 (Isoform 879 RON-1) 2)
S2) RON (isoform 908 GLP-3 (GPC3) 939
MUC1 (Isoform 880 RON-2) (isoform 1)
M6) RON (isoform 909 GLP-3 (GPC3) 940
MUC1 (Isoform 881 RON-3) (isoform 2)
ZD) RON (isoform 910 GLP-3 (GPC3) 941
MUC1 (Isoform 882 RON-4) (isoform 3)
T10) LY6E 911 CLDN6 942
MUC1 (Isoform 883 FRA1 (isoform 1) 912 CLDN16 943
E2) FRA1 (isoform 2) 913 UPK1 B 944
MUC1 (Isoform 884 PSMA (isoform 1) 914 VTCN1 945
J13) PSMA (isoform 915 STRA6 (isoform 1) 946
MUC 16 885 PMSA-3) STRA6 (isoform 2) 947
49
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Antigen SEQ ID Antigen SEQ ID Antigen
SEQ ID
NO: NO:
NO:
STRA6 (isoform 3) 948 EGF (isoform 1) 1070 Vascular 1092
STRA6 (isoform 4) 949 EGF (isoform 2) 1071 endothelial growth
STRA6 (isoform 5) 950 EGF (isoform 3) 1072 factor (isoform
STRA6 (isoform 6 951 IL-2 Receptor 1073 VEGF111)
CD273 (isoform 1) 952 (gamma subunit) Vascular 1093
CD273 (isoform 2) 953 (isoform 1) endothelial growth
CD273 (isoform 3) 954 IL-2 Receptor 1074 factor (isoform L-
PD-L1 (isoform 1) 955 (gamma subunit) VEGF165)
PD-Li (isoform 2) 956 (isoform 2) Vascular
1094
PD-Li (isoform 3) 957 IL-2 Receptor 1075
endothelial growth
CD5 1030 (alpha subunit) factor (isoform L-
CD19 (isoform 1) 1031 Tenascin (isoform 1076
VEGF121)
CD19 (isoform 2) 1032 1) Vascular
1095
CD20 (isoform 1) 1033 Tenascin (isoform 1077
endothelial growth
CD20 (isoform 2) 1034 2) factor (isoform L-
CD25 1035 Tenascin (isoform 1078 VEGF189)
CD37 (isoform 1) 1036 3) Vascular
1096
CD37 (isoform 2) 1037 Tenascin (isoform 1079
endothelial growth
CD37 (isoform 3) 1038 4) factor (isoform L-
Tenascin (isoform 1080 VEGF206)
CD30 (isoform 1) 1039
CD30 (isoform 2) 1040 5) Vascular
1097
Tenascin (isoform 1081 endothelial growth
CD30 (isoform 3) 1041
6) factor (isoform 15)
CD33 (isoform 1) 1042
Endosialin (isoform 1082 Vascular 1098
CD33 (isoform 2) 1043
1) endothelial growth
CD33 (isoform 3) 1044 factor (isoform
16)
Endosialin (isoform 1083
CD45 (isoform 1) 1045 2) Vascular
1099
CD45 (isoform 2) 1046 Vascular 1084 endothelial
growth
CAMPTH-1 1047 endothelial growth factor (isoform 17)
BCMA (isoform 1) 1048 Vascular 1100
factor (isoform
BCMA (isoform 2) 1049 VEGF206) endothelial growth
CS-1 (isoform 1) 1050 Vascular 1085 factor (isoform
18)
CS-1 (isoform 2) 1051 endothelial growth
Integrin alpha V 1101
CS-1 (isoform 3) 1052 factor (isoform
(isoform 1)
B7-H3 (isoform 1) 1053 VEGF189) Integrin
alpha V 1102
B7-H3 (isoform 2) 1054 Vascular 1086 (isoform 2)
B7-H3 (isoform 3) 1055 endothelial growth
Integrin alpha V 1103
B7-H3 (isoform 4) 1056 factor (isoform
(isoform 3)
B7-DC (isoform 1) 1057 VEGF183) Integrin beta 3 1104
B7-DC (isoform 2) 1058 Vascular 1087 (isoform Beta-3A)
B7-DC (isoform 3) 1059 endothelial growth
Integrin beta 3 1105
factor (isoform (isoform Beta-3B)
HLA-DR (isoform 1060 VEGF165) Integrin beta 3 1106
1) Vascular 1088
(isoform Beta-3C)
HLA-DR (isoform 1061 endothelial growth WT1 (isoform 1) 1107
2) factor
(isoform WT1 (isoform 2) 1108
HLA-DR (isoform 1062 VEGF148) WT1 (isoform 3) 1109
3) Vascular 1089
WT1 (isoform 4) 1110
MUC15 (isoform 1063 endothelial growth WT1 (isoform 6) 1111
1) factor
(isoform WT1 (isoform 7) 1112
MUC15 (isoform 1064 VEGF145) WT1 (isoform 8) 1113
2) Vascular 1090
WT1 (isoform 9) 1114
Folate-Binding 1065 endothelial growth LMP2 (isoform 1115
Protein factor (isoform LMP2.L)
A33 1066 VEGF165B) LMP2 (isoform 1116
G250 1067 Vascular 1091 LMP2.S)
Ferritin light chain 1068 endothelial growth
HPV E6 (strain 16) 1117
Ferritin heavy 1069 factor (isoform HPV E6 (strain 18) 1118
chain VEGF121) HPV E7 (strain 16) 1119
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Antigen SEQ ID Antigen SEQ ID Antigen
SEQ ID
NO: NO:
NO:
HPV E7 (strain 18) 1120 Survivin (isoform 1153 PAP
(isoform 1) 1172
P53 nonmutant 1121 4) PAP (isoform 2) 1173
(isoform 1) Survivin (isoform 1154 PAP
(isoform 3) 1174
P53 nonmutant 1122 5) ML-IAP (isoform 1175
(isoform 2) Survivin (isoform 1155 2)
P53 nonmutant 1123 6) ML-IAP (isoform 1176
(isoform 3) Survivin (isoform 1156 1)
P53 nonmutant 1124 7) AFP 1177
(isoform 4) PSA (isoform 1) 1157 ERG (isoform 1178
P53 nonmutant 1125 PSA (isoform 2) 1158 ERG-3)
(isoform 5) PSA (isoform 3) 1159 ERG (isoform 1179
P53 nonmutant 1126 PSA (isoform 4) 1160 ERG-2)
(isoform 6) PSA (isoform 5) 1161 ERG (isoform 1180
P53 nonmutant 1127 hTERT (isoform 1) 1162 ERG-1)
(isoform 7) hTERT (isoform 2) 1163 ERG (isoform 5) 1181
P53 nonmutant 1128 hTERT (isoform 3) 1164 ERG (isoform 7) 1182
(isoform 8) hTERT (isoform 4) 1165 ERG (isoform 8) 1183
P53 nonmutant 1129 Sarcoma 1166 NA17 1184
(isoform 9) translocation PAX3 (isoform 1185
MalenA/MART1 1130 breakpoint fusion Pax3)
Gp100 (isoform 1) 1131 protein (Ewing PAX3
(isoform 1186
Gp100 (isoform 2) 1132 sarcome breakpoint
Pax3A)
Gp100 (isoform 3) 1133 region 1 protein)
PAX3 (isoform 1187
Gp100 (isoform 4) 1134 (isoform EWS) -- Pax3B)
Gp100 (isoform 5) 1135 Sarcoma 1167 PAX3
(isoform 1188
PR1 (TMEM37) 1136 translocation Pax3G)
BCR-abl (isofrom 1137 breakpoint fusion
PAX3 (isoform 1189
X3) protein (Ewing Pax3H)
BCR-abl (isofrom 1138 sarcome breakpoint
PAX3 (isoform 6) 1190
Y5) region 1 protein) PAX3 (isoform 7)
1191
BCR-abl (isofrom 1139 (isoform EWS-B) PAX3
(isoform 1192
X9) Sarcoma 1168 Pax3E)
BCR-abl (isofrom 1140 translocation ALK
1193
X2) breakpoint fusion Androgen receptor
1194
BCR-abl (isofrom 1141 protein (Ewing
(isoform 1)
e8a2 variant) sarcome breakpoint Androgen receptor
1195
BCR-abl (isofrom 1142 region 1 protein)
(isoform 2)
Y3) (isoform
3) Androgen receptor 1196
BCR-abl (isofrom 1143 Sarcoma 1169 (isoform 3)
Y2) translocation
Androgen receptor 1197
BCR-abl (isofrom 1144 breakpoint fusion
(isoform 4)
Y1) protein (Ewing
Cyclin B1 (isoform 1198
BCR-abl (isofrom 1145 sarcome breakpoint
1)
X6) region 1 protein)
Cyclin B1 (isoform 1199
BCR-abl (isofrom 1146 (isoform 4) 2)
Y4) Sarcoma 1170
MYCN 1200
translocation
BCR-abl (isofrom 1147 RhoC
1201
breakpoint fusion
Y6) TRP-2 1202
protein (Ewing
Tyrosinase 1148 TRP-2 1203
sarcome breakpoint
(isoform 1) TRP-2 1204
region 1 protein)
Tyrosinase 1149 PSCA 1205
(isoform 5)
(isoform 2) MAGE Al 1206
Sarcoma 1171
Survivin (isoform 1150 CYP1B1
1207
translocation
1) breakpoint fusion BORIS (isoform 1) 1208
Survivin (isoform 1151 protein (Ewing BORIS
(isoform 2) 1209
2)
sarcome breakpoint BORIS (isoform 3) 1210
Survivin (isoform 1152
region 1 protein) BORIS (isoform 4) 1211
3) (isoform 6) BORIS (isoform 5) 1212
51
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Antigen SEQ ID Antigen SEQ ID Antigen
SEQ ID
NO: NO:
NO:
BORIS (isoform 6) 1213 Legumain (isoform 1252 1)
BORIS (isoform 7) 1214 1) VEGFR1 (isoform 1278
BORIS (isoform 8) 1215 Legumain (isoform 1253 2)
BORIS (isoform 9) 1216 2) VEGFR1 (isoform 1279
BORIS (isoform 1217 Legumain (isoform 1254 3)
10) 3)
VEGFR1 (isoform 1280
BORIS (isoform 1218 VEGFR2 (isoform 1255 4)
11) 1)
VEGFR1 (isoform 1281
ETV6-AML 1219 VEGFR2 (isoform 1256 5)
NY-BR-1 1220 2) VEGFR1 (isoform 1282
RGS5 (isoform 1) 1221 VEGFR2 (isoform 1257 6)
RGS5 (isoform 2) 1222 3) VEGFR1 (isoform
1283
RGS5 (isoform 3) 1223 MAD-CT-1 1258 7)
SART3 (isoform 1) 1224 (isoform 1) VEGFR1 (isoform 1284
SART3 (isoform 2) 1225 MAD-CT-1 1259 8)
SART3 (isoform 3) 1226 (isoform 2) Endoglin (isoform
1285
SART3 (isoform 4) 1227 PDGFR-B (isoform 1260 long)
Carbonic anhydrase 1228 1) Endoglin (isoform
1286
IX PDGFR-B (isoform 1261 short)
PAX5 (isoform 1) 1229 2) VISTA
1287
PAX5 (isoform 2) 1230 ROR2 1262 MAGE C2
1288
PAX5 (isoform 3) 1231 TMPRSS3 1263 MAGE A4
1289
PAX5 (isoform 4) 1232 (isoform A) GAGE
2B/C 1290
PAX5 (isoform 5) 1233 TMPRSS3 1264 GAGE 7
1291
PAX5 (isoform 6) 1234 (isoform B) GAGE
12B/C/D/E 1292
TMPRSS3 1265 GAGE 2D 1293
PAX5 (isoform 7) 1235
PAX5 (isoform 8) 1236 (isoform D) GAGE 1
1294
TMPRSS3 1266 GAGE 5 1295
PAX5 (isoform 9) 1237
(isoform T) GAGE 121 1296
PAX5 (isoform 10) 1238
TMPRSS3 1267 GAGE 12F 1297
PAX5 (isoform 11) 1239
(isoform E) GAGE 13 1298
0Y-TES1 1240
TMPRSS3 1268 GAGE 12J 1299
Sperm protein 17 1241
(isoform 6) GAGE 2A 1300
LCK (isoform 1242
TMPRSS4 1269
long) GAGE 6 1301
(isoform 1)
LCK (isoform 1243 GAGE 10 1302
TMPRSS4 1270
short) GAGE 4 1303
(isoform 2)
LCK (isoform 3) 1244 GAGE 12G 1304
TMPRSS4 1271
HMWMAA 1245 GAGE 12H 1305
(isoform 3)
AKAP-4 (isoform 1246 GAGE 2E 1306
TMPRSS4 1272
1) TRAIL1 (isoform 1307
(isoform 4)
AKAP-4 (isoform 1247 long)
TMEM238 1273
2) TRAIL (isoform 3) 1308
C10RF186 1274
55X2 (isoform 1) 1248 TRAIL
(isoform 1309
Fos-related antigen 1275
55X2 (isoform 2) 1249 short)
1 (isoform 1)
XAGE 1 (isoform 1250 PAGE4 1310
Fos-related antigen 1276
B)
1 (isoform 2)
XAGE 1 (isoform 1251
VEGFR1 (isoform 1277
D)
[0168] In some embodiments, an amino acid sequence of the tumor antigen has at
least 80%
sequence identity with the amino acid sequence of a tumor antigen selected
from the group
consisting of HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), EGFR, fibroblast
activation protein
(FAP), tenascin, a metalloproteinase, endosialin, vascular endothelial growth
factor, avf33, WT1,
LMP2, HPV E6, HPV E7, Her-2/neu, p53 nonmutant, NY-ESO-1, GLP-3, MelanA/MART1,
Ras
52
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a
Sarcoma
translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17,
PAX3, ALK,
androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, mesothelin (MSLN), PSCA, MAGE
Al,
MAGE-A3, CYP1B1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, Carbonic
anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17, LCK, MAGE C2, MAGE A4, GAGE,
TRAILl, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4,VEGFR2,
MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EPCAM, CA6, NAPI2B, TROP2,
Claudin-6 (CLDN6), Claudin-16 (CLDN16), CLDN18.2, RON, LY6E, FRA, DLL3, PTK7,
Uroplakin-1B (UPK1B), LIV1, ROR1, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186,
Fos-related antigen 1, VEGFR1, endoglin, PD-L1, VTCN1 (B7-H4), VISTA, or a
fragment
thereof, and a fragment thereof. In some embodiments, an amino acid sequence
of the tumor
antigen has at least 80% sequence identity with the amino acid sequence of a
tumor antigen
selected from TABLE 1.
[0169] In some embodiments, an amino acid sequence of the tumor antigen has at
least 80%
sequence identity with the amino acid sequence of a tumor antigen selected
from the group
consisting of HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B,
TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7,
LIV1, ROR1, MAGE-A3, NY-ES0-1, Endoglin, CD204, CD206, CD301, VTCN1, VISTA,
GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, and
LRRC15, but not HER2 when the second binding domain specifically binds to
CD40.
[0170] A binding domain of a conjugate or antibody construct can be selected
from any domain
that binds to an antigen including, but not limited to, from a monoclonal
antibody, a polyclonal
antibody, a recombinant antibody, or a functional fragment thereof, for
example, a heavy chain
variable domain (VH) and a light chain variable domain (VL), or from a non-
antibody scaffold,
such as a DARPin, an affimer, an avimer, a knottin, a monobody, lipocalin, an
anticalin, 'T-
body', an affibody, a peptibody, an affinity clamp, an ectodomain, a receptor
ectodomain, a
receptor, a cytokine, a ligand, an immunocytokine, a centryin, a T-cell
receptor, or a recombinant
T-cell receptor. The antigen binding domain of a conjugate or antibody
construct can be at least
80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to
an antigen binding
domain selected from, but not limited to, a monoclonal antibody, a polyclonal
antibody, a
recombinant antibody, or a functional fragment thereof, for example, a heavy
chain variable
domain (VH) and a light chain variable domain (VL), or a DARPin, an affimer,
an avimer, a
knottin, a monobody, a lipocalin, an anticalin, 'T-body', an affibody, a
peptibody, an affinity
53
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand,
an
immunocytokine, a centryin, a T-cell receptor, or a recombinant T-cell
receptor.
[0171] A binding domain of a conjugate or antibody construct, for example an
antigen binding
domain from a monoclonal antibody, can comprise a light chain and a heavy
chain. In one aspect,
the monoclonal antibody binds to an antigen present on the surface of an
immune cell (immune
cell antigen) and comprises the light chain of an anti-immune cell antigen
antibody and the heavy
chain of an anti-immune cell antigen antibody, which bind to an immune cell
antigen. In another
aspect, the monoclonal antibody binds to an antigen present on the surface of
an antigen
presenting cell (APC antigen) and comprises the light chain of an anti-APC
antigen antibody and
the heavy chain of an anti-APC antigen antibody, which bind to an APC antigen.
In another
aspect, the monoclonal antibody binds to CD40 and comprises the light chain of
an anti-CD40
antibody and the heavy chain of an anti-CD40 antibody, which bind to a CD40
antigen. In
another aspect, the monoclonal antibody binds to a tumor antigen comprises the
light chain of a
tumor antigen antibody and the heavy chain of a tumor antigen antibody, which
bind to the tumor
antigen.
[0172] A conjugate or antibody construct can comprise an antibody. An antibody
molecule can
consist of two identical light protein chains (light chains) and two identical
heavy protein chains
(heavy chains), all held together covalently by precisely located disulfide
linkages. The N-
terminal regions of the light and heavy chains together can form the antigen
recognition site of
each antibody. Structurally, various functions of an antibody can be confined
to discrete protein
domains (i.e., regions). The sites that can recognize and can bind to antigen
consist of three
complementarity determining regions (CDRs) that can lie within the variable
heavy chain regions
and variable light chain regions at the N-terminal ends of the two heavy and
two light chains. The
constant domains can provide the general framework of the antibody and may not
be involved
directly in binding the antibody to an antigen, but can be involved in various
effector functions,
such as participation of the antibody in antibody-dependent cellular
cytotoxicity (ADCC).
[0173] The domains of natural light chain variable regions and heavy chain
variable regions can
have the same general structures, and each domain can comprise four framework
regions, whose
sequences can be somewhat conserved, connected by three hyper-variable regions
or CDRs. The
four framework regions can largely adopt a 13-sheet conformation and the CDRs
can form loops
connecting, and in some aspects forming part of, the 13 -sheet structure. The
CDRs in each chain
can be held in close proximity by the framework regions and, with the CDRs
from the other
chain, can contribute to the formation of the antigen binding site.
54
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0174] An antibody of a conjugate or antibody construct can comprise an
antibody of any type,
which can be assigned to different classes of immunoglobins, e.g., IgA, IgD,
IgE, IgG, and IgM.
Several different classes can be further divided into isotypes, e.g., IgGl,
IgG2, IgG3, IgG4, IgAl,
and IgA2. An antibody can further comprise a light chain and a heavy chain,
often more than one
chain. The heavy-chain constant regions (Fc) that corresponds to the different
classes of
immunoglobulins can be a, 6, , y, and 11, respectively. The light chains can
be one of either
kappa or lc and lambda or k, based on the amino acid sequences of the constant
domains. The Fc
region can comprise an Fc domain. An Fc receptor can bind to an Fc domain. A
conjugate can
also comprise any fragment or recombinant form thereof, including but not
limited to an scFv,
Fab, variable Fc fragment, domain antibody, and any other fragment thereof
that can specifically
bind to an antigen.
[0175] An antibody can comprise an antigen binding domain which can refer to a
portion of an
antibody comprising the antigen recognition portion, i.e., an antigenic
determining variable
region of an antibody sufficient to confer recognition and binding of the
antigen recognition
portion to a target, such as an antigen, i.e., the epitope. Examples of
antibody binding domains
can include, but are not limited to, Fab, variable Fv fragment and other
fragments, combinations
of fragments or types of fragments known or knowable to one of ordinary skill
in the art.
[0176] A conjugate or antibody construct can comprise an antigen binding
domain of an
antibody. An antigen binding domain of an antibody can comprise one or more
light chain (LC)
CDRs (LCDRs) and one or more heavy chain (HC) CDRs (HCDRs), one or more LCDRs
or one
or more HCDRs. For example, an antibody binding domain of an antibody can
comprise one or
more of the following: a light chain complementary determining region 1
(LCDR1), a light chain
complementary determining region 2 (LCDR2), or a light chain complementary
determining
region 3 (LCDR3). For another example, an antibody binding domain can comprise
one or more
of the following: a heavy chain complementary determining region 1 (HCDR1), a
heavy chain
complementary determining region 2 (HCDR2), or a heavy chain complementary
determining
region 3 (HCDR3). In some embodiments an antibody binding domain comprises all
of the
following: a light chain complementary determining region 1 (LCDR1), a light
chain
complementary determining region 2 (LCDR2), a light chain complementary
determining region
3 (LCDR3), a heavy chain complementary determining region 1 (HCDR1), a heavy
chain
complementary determining region 2 (HCDR2), and a heavy chain complementary
determining
region 3 (HCDR3). Unless stated otherwise, the CDRs described herein can be
defined according
to the IMGT (the international ImMunoGeneTics information system). An antigen
binding
domain can comprise only the heavy chain of an antibody (e.g., does not
include any other
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
portion of the antibody). An antigen binding domain can comprise only the
variable domain of
the heavy chain of an antibody. Alternatively, an antigen binding domain can
comprise only the
light chain of an antibody. An antigen binding domain can comprise only the
variable light chain
of an antibody.
[0177] A conjugate or antibody construct can comprise an antibody fragment. An
antibody
fragment can include (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two
Fab fragments
linked by a disulfide bridge at the hinge region; and (iii) a Fv fragment
consisting of the VL and
VH domains of a single arm of an antibody. Although the two domains of the Fv
fragment, VL
and VH, can be coded for by separate genes, they can be linked by a synthetic
linker to be made
as a single protein chain in which the VL and VH regions pair to form
monovalent molecules.
[0178] F(ab')2 and Fab' moieties can be produced, for example, recombinantly
or by treating
immunoglobulin (monoclonal antibody) with a protease such as pepsin and
papain, and can
include an antibody fragment generated by digesting immunoglobulin near the
disulfide bonds
existing between the hinge regions in each of the two H chains. The Fab
fragment can also
contain the constant domain of the light chain and the first constant domain
(CH1) of the heavy
chain. Fab' fragments can differ from Fab fragments by the addition of a few
residues at the
carboxyl terminus of the heavy chain CH1 domain including one or more
cysteine(s) from the
antibody hinge region.
[0179] An Fv can be the minimum antibody fragment which contains a complete
antigen-
recognition and antigen-binding site. This region can consist of a dimer of
one heavy chain and
one light chain variable domain in tight, non-covalent association. In this
configuration, the three
CDRs of each variable domain can interact to define an antigen-binding site on
the surface of the
VH-VL dimer. A single variable domain (or half of an Fv comprising only three
CDRs specific
for an antigen) can recognize and bind to antigen, although at a lower
affinity than the entire
binding site.
[0180] An antibody used herein can be "humanized." Humanized forms of non-
human (e.g.,
murine) antibodies can be chimeric immunoglobulins, immunoglobulin chains or
fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of
antibodies), which
can contain minimal sequences derived from non-human immunoglobulin. In
general, the
humanized antibody can comprise substantially all of at least one, and
typically two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-human
immunoglobulin and all or substantially all of the framework (FR) regions are
those of a human
immunoglobulin sequence. The humanized antibody can also comprise at least a
portion of an
56
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
immunoglobulin constant region (Fc), typically that of a human immunoglobulin
consensus
sequence.
[0181] An antibody described herein can be a human antibody. As used herein,
"human
antibodies" can include antibodies having, for example, the amino acid
sequence of a human
immunoglobulin and include antibodies isolated from human immunoglobulin
libraries or from
animals transgenic for one or more human immunoglobulins that do not express
endogenous
immunoglobulins. Human antibodies can be produced using transgenic mice which
are incapable
of expressing functional endogenous immunoglobulins, but which can express
human
immunoglobulin genes. Completely human antibodies that recognize a selected
epitope can be
generated using guided selection. In this approach, a selected non-human
monoclonal antibody,
e.g., a mouse antibody, is used to guide the selection of a completely human
antibody
recognizing the same epitope
[0182] An antibody described herein can be a bispecific antibody or a dual
variable domain
antibody (DVD). Bispecific and DVD antibodies are monoclonal, often human or
humanized,
antibodies that have binding specificities for at least two different
antigens.
[0183] An antibody described herein can be a derivatized antibody. For
example, derivatized
antibodies can be modified by glycosylation, acetylation, pegylation,
phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic cleavage, or
the like.
[0184] An antibody described herein can have a sequence that has been modified
to alter at least
one constant region-mediated biological effector function relative to the
corresponding wild type
sequence. For example, in some embodiments, the antibody can be modified to
reduce at least
one constant region-mediated biological effector function relative to an
unmodified antibody, e.g.,
reduced or increased binding to an Fc receptor (FcR). FcR binding can be
reduced or increased
by, for example, mutating the immunoglobulin constant region segment of the
antibody at
particular regions necessary for FcR interactions.
[0185] An antibody described herein can be modified to acquire or improve at
least one constant
region-mediated biological effector function relative to an unmodified
antibody, e.g., to enhance
FcyR interactions. For example, an antibody with a constant region that binds
FcyRIIA, FcyRIIB
and/or FcyRIIIA with greater affinity than the corresponding wild type
constant region can be
produced according to the methods described herein.
[0186] An antibody described herein can bind to tumor cells, such as an
antibody against a cell
surface receptor or a tumor antigen.
[0187] A conjugate or antibody construct can comprise a first binding domain.
A conjugate or
antibody construct can comprise a first binding domain that specifically binds
to an antigen. A
57
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
conjugate or antibody construct can comprise a first binding domain that
specifically binds to a
tumor antigen. A first binding domain can specifically bind to a tumor
antigen, wherein the
tumor antigen has an amino acid sequence that comprises at least 80% homology
to an amino
acid sequence of an antigen selected from the group consisting of HER2, EGFR,
CMET, HER3,
MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP,
EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, and a
fragment thereof. A first binding domain can specifically bind to a tumor
antigen, wherein the
tumor antigen has an amino acid sequence that comprises at least 80% homology
to an amino
acid sequence of an antigen selected from the group consisting of EGFR, CMET,
HER3, MUC1,
MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2,
RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, LRRC15, GLP-
3, CLDN6, CLDN16, UPK1B, VTCN1 (B7-H4) and STRA6 and a fragment thereof. A
first
binding domain can specifically bind to a tumor antigen, wherein the tumor
antigen has an amino
acid sequence that comprises at least 80% homology to an amino acid sequence
of an antigen
selected from the group consisting of HER2, EGFR, CMET, HER3, MUC1, MUC16,
EPCAM,
MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA,
PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, LRRC15, GLP-3, CLDN6,
CLDN16, UPK1B, VTCN1 (B7-H4) and STRA6 and a fragment thereof. A conjugate can
comprise a first binding domain that specifically binds to a tumor antigen on
a tumor cell, to an
immune cell such as an antigen presenting cell, to an immune cell other than
an antigen
presenting cell or to an antigen presenting cell.
[0188] A conjugate or antibody construct can comprise a first binding domain
that specifically
binds to a tumor antigen. A conjugate or antibody construct can comprise a
first binding domain
comprising one or more CDRs. A first binding domain can comprise at least 80%
sequence
identity to any sequence in TABLE 3. A first binding domain can comprise at
least 80%
sequence identity to any sequence in TABLE 3 or TABLE 4. A conjugate can
comprise a first
binding domain that binds to a tumor antigen, wherein the first binding domain
comprises at least
80% sequence identity to: a) HCDR1 comprising an amino acid sequence of SEQ ID
NO: 13,
HCDR2 comprising an amino acid sequence of SEQ ID NO: 14, HCDR3 comprising an
amino
acid sequence of SEQ ID NO: 15, LCDR1 comprising an amino acid sequence of SEQ
ID NO:
18, LCDR2 comprising an amino acid sequence of SEQ ID NO: 19, and LCDR3
comprising an
amino acid sequence of SEQ ID NO: 20; b) HCDR1 comprising an amino acid
sequence of SEQ
ID NO: 26, HCDR2 comprising an amino acid sequence of SEQ ID NO: 27, HCDR3
comprising
an amino acid sequence of SEQ ID NO: 28, LCDR1 comprising an amino acid
sequence of SEQ
58
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
ID NO: 31, LCDR2 comprising an amino acid sequence of SEQ ID NO: 32, and LCDR3
comprising an amino acid sequence of SEQ ID NO: 33; c) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 39, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 40,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 41, LCDR1 comprising an
amino
acid sequence of SEQ ID NO: 44, LCDR2 comprising an amino acid sequence of SEQ
ID NO:
45, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 46; d) HCDR1
comprising
an amino acid sequence of SEQ ID NO: 52, HCDR2 comprising an amino acid
sequence of SEQ
ID NO: 53, HCDR3 comprising an amino acid sequence of SEQ ID NO: 54, LCDR1
comprising
an amino acid sequence of SEQ ID NO: 57, LCDR2 comprising an amino acid
sequence of SEQ
ID NO: 58, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 59; e)
HCDR1
comprising an amino acid sequence of SEQ ID NO: 65, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 66, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 67,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 70, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 71, and LCDR3 comprising an amino acid sequence of
SEQ ID
NO: 72; f) HCDR1 comprising an amino acid sequence of SEQ ID NO: 78, HCDR2
comprising
an amino acid sequence of SEQ ID NO: 79, HCDR3 comprising an amino acid
sequence of SEQ
ID NO: 80, LCDR1 comprising an amino acid sequence of SEQ ID NO: 83, LCDR2
comprising
an amino acid sequence of SEQ ID NO: 84, and LCDR3 comprising an amino acid
sequence of
SEQ ID NO: 85; g) HCDR1 comprising an amino acid sequence of SEQ ID NO: 91,
HCDR2
comprising an amino acid sequence of SEQ ID NO: 92, HCDR3 comprising an amino
acid
sequence of SEQ ID NO: 93, LCDR1 comprising an amino acid sequence of SEQ ID
NO: 96,
LCDR2 comprising an amino acid sequence of SEQ ID NO: 97, andLCDR3 comprising
an
amino acid sequence of SEQ ID NO: 98; h) HCDR1 comprising an amino acid
sequence of SEQ
ID NO: 104, HCDR2 comprising an amino acid sequence of SEQ ID NO: 105, HCDR3
comprising an amino acid sequence of SEQ ID NO: 106, LCDR1 comprising an amino
acid
sequence of SEQ ID NO: 109, LCDR2 comprising an amino acid sequence of SEQ ID
NO: 110,
and LCDR3 comprising an amino acid sequence of SEQ ID NO: 111; i) HCDR1
comprising an
amino acid sequence of SEQ ID NO: 117, HCDR2 comprising an amino acid sequence
of SEQ
ID NO: 118, HCDR3 comprising an amino acid sequence of SEQ ID NO: 119, LCDR1
comprising an amino acid sequence of SEQ ID NO: 122, LCDR2 comprising an amino
acid
sequence of SEQ ID NO: 123, andLCDR3 comprising an amino acid sequence of SEQ
ID NO:
124; j) HCDR1 comprising an amino acid sequence of SEQ ID NO: 130, HCDR2
comprising an
amino acid sequence of SEQ ID NO: 131, HCDR3 comprising an amino acid sequence
of SEQ
ID NO: 132, LCDR1 comprising an amino acid sequence of SEQ ID NO: 135, LCDR2
59
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprising an amino acid sequence of SEQ ID NO: 136, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 137; k) HCDR1 comprising an amino acid sequence of SEQ
ID NO:
143, HCDR2 comprising an amino acid sequence of SEQ ID NO: 144, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 145, LCDR1 comprising an amino acid sequence
of SEQ
ID NO: 148, LCDR2 comprising an amino acid sequence of SEQ ID NO: 149, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 150; 1) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 156, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 157,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 158, LCDR1 comprising an
amino
acid sequence of SEQID NO: 161, LCDR2 comprising an amino acid sequence of SEQ
ID NO:
162, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 163; m) HCDR1
comprising an amino acid sequence of SEQ ID NO: 169, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 170, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 171,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 174, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 175, and LCDR3 comprising an amino acid sequence
of SEQ ID
NO: 176; n) HCDR1 comprising an amino acid sequence of SEQ ID NO: 182, HCDR2
comprising an amino acid sequence of SEQ ID NO: 183, HCDR3 comprising an amino
acid
sequence of SEQ ID NO: 184, LCDR1 comprising an amino acid sequence of SEQ ID
NO: 187,
LCDR2 comprising an amino acid sequence of SEQ ID NO: 188, and LCDR3
comprising an
amino acid sequence of SEQ ID NO: 189; o) HCDR1 comprising an amino acid
sequence of
SEQ ID NO: 195, HCDR2 comprising an amino acid sequence of SEQ ID NO: 196,
HCDR3
comprising an amino acid sequence of SEQ ID NO: 197, LCDR1 comprising an amino
acid
sequence of SEQ ID NO: 200, LCDR2 comprising an amino acid sequence of SEQ ID
NO: 201,
and LCDR3 comprising an amino acid sequence of SEQ ID NO: 202; p) HCDR1
comprising an
amino acid sequence of SEQ ID NO: 208, HCDR2 comprising an amino acid sequence
of SEQ
ID NO: 209, HCDR3 comprising an amino acid sequence of SEQ ID NO: 210, LCDR1
comprising an amino acid sequence of SEQ ID NO: 213, LCDR2 comprising an amino
acid
sequence of SEQ ID NO: 214, and LCDR3 comprising an amino acid sequence of SEQ
ID NO:
215; q) HCDR1 comprising an amino acid sequence of SEQ ID NO: 805, HCDR2
comprising an
amino acid sequence of SEQ ID NO: 806, HCDR3 comprising an amino acid sequence
of SEQ
ID NO: 807, LCDR1 comprising an amino acid sequence of SEQ ID NO: 808, LCDR2
comprising an amino acid sequence of SEQ ID NO: 809, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 810; r) HCDR1 comprising an amino acid sequence of SEQ
ID NO:
823, HCDR2 comprising an amino acid sequence of SEQ ID NO: 824, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 825, LCDR1 comprising an amino acid sequence
of SEQ
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
ID NO: 826, LCDR2 comprising an amino acid sequence of SEQ ID NO: 827, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 828; s) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 221, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 222,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 223, LCDR1 comprising an
amino
acid sequence of SEQ ID NO: 226, LCDR2 comprising an amino acid sequence of
SEQ ID NO:
227, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 228; t) HCDR1
comprising
an amino acid sequence of SEQ ID NO: 260, HCDR2 comprising an amino acid
sequence of
SEQ ID NO: 261, HCDR3 comprising an amino acid sequence of SEQ ID NO: 262,
LCDR1
comprising an amino acid sequence of SEQ ID NO: 265, LCDR2 comprising an amino
acid
sequence of SEQ ID NO: 266, and LCDR3 comprising an amino acid sequence of SEQ
ID NO:
267; u) HCDR1 comprising an amino acid sequence of SEQ ID NO: 273, HCDR2
comprising an
amino acid sequence of SEQ ID NO: 274, HCDR3 comprising an amino acid sequence
of SEQ
ID NO: 275, LCDR1 comprising an amino acid sequence of SEQ ID NO: 278, LCDR2
comprising an amino acid sequence of SEQ ID NO: 279, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 280; v) HCDR1 comprising an amino acid sequence of SEQ
ID NO:
286, HCDR2 comprising an amino acid sequence of SEQ ID NO: 287, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 288, LCDR1 comprising an amino acid sequence
of SEQ
ID NO: 291, LCDR2 comprising an amino acid sequence of SEQ ID NO: 292, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 293; w) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 299, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 300,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 301, LCDR1 comprising an
amino
acid sequence of SEQ ID NO: 304, LCDR2 comprising an amino acid sequence of
SEQ ID NO:
305, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 306; x) HCDR1
comprising an amino acid sequence of SEQ ID NO: 312, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 313, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 314,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 317, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 318, and LCDR3 comprising an amino acid sequence
of SEQ ID
NO: 319; y) HCDR1 comprising an amino acid sequence of SEQ ID NO: 325, HCDR2
comprising an amino acid sequence of SEQ ID NO: 326, HCDR3 comprising an amino
acid
sequence of SEQ ID NO: 327, LCDR1 comprising an amino acid sequence of SEQ ID
NO: 330,
LCDR2 comprising an amino acid sequence of SEQ ID NO: 331, and LCDR3
comprising an
amino acid sequence of SEQ ID NO: 332; z) HCDR1 comprising an amino acid
sequence of
SEQ ID NO: 338, HCDR2 comprising an amino acid sequence of SEQ ID NO: 339,
HCDR3
comprising an amino acid sequence of SEQ ID NO: 340, LCDR1 comprising an amino
acid
61
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
sequence of SEQ ID NO: 343, LCDR2 comprising an amino acid sequence of SEQ ID
NO: 344,
and LCDR3 comprising an amino acid sequence of SEQ ID NO: 345; aa) HCDR1
comprising an
amino acid sequence of SEQ ID NO: 351, HCDR2 comprising an amino acid sequence
of SEQ
ID NO: 352, HCDR3 comprising an amino acid sequence of SEQ ID NO: 353, LCDR1
comprising an amino acid sequence of SEQ ID NO: 356, LCDR2 comprising an amino
acid
sequence of SEQ ID NO: 357, and LCDR3 comprising an amino acid sequence of SEQ
ID NO:
358; bb) HCDR1 comprising an amino acid sequence of SEQ ID NO: 364, HCDR2
comprising
an amino acid sequence of SEQ ID NO: 365, HCDR3 comprising an amino acid
sequence of
SEQ ID NO: 366, LCDR1 comprising an amino acid sequence of SEQ ID NO: 369,
LCDR2
comprising an amino acid sequence of SEQ ID NO: 370, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 371; cc) HCDR1 comprising an amino acid sequence of SEQ
ID NO:
377, HCDR2 comprising an amino acid sequence of SEQ ID NO: 378, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 379, LCDR1 comprising an amino acid sequence
of SEQ
ID NO: 382, LCDR2 comprising an amino acid sequence of SEQ ID NO: 383, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 384; dd) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 390, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 391,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 392, LCDR1 comprising an
amino
acid sequence of SEQ ID NO: 395, LCDR2 comprising an amino acid sequence of
SEQ ID NO:
396, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 397; ee) HCDR1
comprising an amino acid sequence of SEQ ID NO: 403, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 404, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 405,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 408, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 409, and LCDR3 comprising an amino acid sequence
of SEQ ID
NO: 410; if) HCDR1 comprising an amino acid sequence of SEQ ID NO: 416, HCDR2
comprising an amino acid sequence of SEQ ID NO: 417, HCDR3 comprising an amino
acid
sequence of SEQ ID NO: 418, LCDR1 comprising an amino acid sequence of SEQ ID
NO: 421,
LCDR2 comprising an amino acid sequence of SEQ ID NO: 422, and LCDR3
comprising an
amino acid sequence of SEQ ID NO: 423; gg) HCDR1 comprising an amino acid
sequence of
SEQ ID NO: 429, HCDR2 comprising an amino acid sequence of SEQ ID NO: 430,
HCDR3
comprising an amino acid sequence of SEQ ID NO: 431, LCDR1 comprising an amino
acid
sequence of SEQ ID NO: 434, LCDR2 comprising an amino acid sequence of SEQ ID
NO: 435,
and LCDR3 comprising an amino acid sequence of SEQ ID NO: 436; hh) HCDR1
comprising an
amino acid sequence of SEQ ID NO: 442, HCDR2 comprising an amino acid sequence
of SEQ
ID NO: 443, HCDR3 comprising an amino acid sequence of SEQ ID NO: 444, LCDR1
62
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprising an amino acid sequence of SEQ ID NO: 447, LCDR2 comprising an amino
acid
sequence of SEQ ID NO: 448, and LCDR3 comprising an amino acid sequence of SEQ
ID NO:
449; ii) HCDR1 comprising an amino acid sequence of SEQ ID NO: 455, HCDR2
comprising an
amino acid sequence of SEQ ID NO: 456, HCDR3 comprising an amino acid sequence
of SEQ
ID NO: 457, LCDR1 comprising an amino acid sequence of SEQ ID NO: 460, LCDR2
comprising an amino acid sequence of SEQ ID NO: 461, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 462; jj) HCDR1 comprising an amino acid sequence of SEQ
ID NO:
468, HCDR2 comprising an amino acid sequence of SEQ ID NO: 469, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 470, LCDR1 comprising an amino acid sequence
of SEQ
ID NO: 473, LCDR2 comprising an amino acid sequence of SEQ ID NO: 474, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 475; kk) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 481, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 482,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 483, LCDR1 comprising an
amino
acid sequence of SEQ ID NO: 486, LCDR2 comprising an amino acid sequence of
SEQ ID NO:
487, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 488;11) HCDR1
comprising an amino acid sequence of SEQ ID NO: 494, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 495, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 496,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 499, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 500, and LCDR3 comprising an amino acid sequence
of SEQ ID
NO: 501; or mm) HCDR1 comprising an amino acid sequence of SEQ ID NO: 673,
HCDR2
comprising an amino acid sequence of SEQ ID NO: 674, HCDR3 comprising an amino
acid
sequence of SEQ ID NO: 675, LCDR1 comprising an amino acid sequence of SEQ ID
NO: 676,
LCDR2 comprising an amino acid sequence of SEQ ID NO: 677, and LCDR3
comprising an
amino acid sequence of SEQ ID NO: 678.
[0189] A conjugate or antibody construct can comprise a first binding domain
that specifically
binds to a tumor antigen. A conjugate can comprise a first binding domain
comprising one or
more variable domains. A conjugate or antibody construct can comprise a first
binding domain
comprising a light chain variable domain (VL domain). A first binding domain
can comprise a
VL sequence in TABLE 5. A first binding domain can comprise at least 80%
sequence identity
to a VL sequence in TABLE 5. A conjugate or antibody construct can comprise a
first binding
domain comprising a heavy chain variable domain (VH domain). A first binding
domain can
comprise VH sequence in TABLE 5. A first binding domain can comprise at least
80% sequence
identity to any VH sequence in TABLE 5. A first binding domain can comprise at
least 80%
sequence identity to a sequence in TABLE 5. A conjugate or antibody construct
can comprise a
63
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
first binding domain comprising a light chain variable domain (VL domain). A
first binding
domain can comprise a VL sequence in TABLE 5 or TABLE 6. A first binding
domain can
comprise at least 80% sequence identity to a VL sequence in TABLE 5 or TABLE
6. A
conjugate or antibody construct can comprise a first binding domain comprising
a heavy chain
variable domain (VH domain). A first binding domain can comprise VH sequence
in TABLE 5
or TABLE 6. A first binding domain can comprise at least 80% sequence identity
to any VH
sequence in TABLE 5 or TABLE 6. A first binding domain can comprise at least
80% sequence
identity to a sequence in TABLE 5 or TABLE 6.
[0190] A conjugate or antibody construct can comprise a first binding domain
that specifically
binds to a tumor antigen, wherein the first binding domain comprises: a) a VH
sequence having
at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 12, and
a VL sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
17; b) a VH
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 25,
and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 30; c) a VH sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 38, and a VL sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 43; d) a VH sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 51, and a VL sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 56; e) a VH sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 64, and a VL
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 69; f) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
77, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 82; g)
a VH sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
90, and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ
ID NO: 95; h) a VH sequence having at least 80% sequence identity to an amino
acid sequence
of SEQ ID NO: 103, and a VL sequence having at least 80% sequence identity to
an amino acid
sequence of SEQ ID NO: 108; i) a VH sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 116, and a VL sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 121; j) a VH sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 129, and a VL
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 134; k) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
142, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 147;
64
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
1) a VH sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 155, and a VL sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 160; m) a VH sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 168, and a VL sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 173; n) a VH sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 181, and a VL sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 186; o) a VH
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 194, and a
VL sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
199; p) a VH
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 207,
and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 212; q) a VH sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 811, and a VL sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 812; r) a VH sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 829, and a VL sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 830; s) a VH sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 220, and a VL
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 225; t) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
259, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 264;
u) a VH sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 272, and a VL sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 277; v) a VH sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 285, and a VL sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 290; w) a VH sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 298, and a VL sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 303; x) a VH
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 311, and a
VL sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
316; y) a VH
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 324,
and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 328; z) a VH sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 337, and a VL sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 342; aa) a VH sequence having at least 80% sequence
identity to an
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
amino acid sequence of SEQ ID NO: 350, and a VL sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 355; bb) a VH sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 363, and a VL
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 368; cc) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
376, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 381;
dd) a VH sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 389, and a VL sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 394; ee) a VH sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 402, and a VL sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 407; ff) a VH sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 415, and a VL sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 420; gg) a VH
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 428, and a
VL sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
433; hh) a VH
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 441,
and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 446; ii) a VH sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 454, and a VL sequence having at least 80% sequence identity to an
amino acid
sequence of SEQ ID NO: 459; jj) a VH sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 467, and a VL sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 472; kk) a VH sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 480, and a VL
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 485;11) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
493, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 498;
or mm) a VH sequence having at least 80% sequence identity to an amino acid
sequence of SEQ
ID NO: 679, and a VL sequence having at least 80% sequence identity to an
amino acid sequence
of SEQ ID NO: 680.
[0191] A conjugate or antibody construct can comprise a first binding domain
and an Fc domain,
wherein the first binding domain and the Fc domain comprise an antibody. A
first binding
domain can bind to a tumor antigen. A conjugate or antibody construct can
comprise an antibody
light chain. A conjugate or antibody construct can comprise a light chain
comprising a light chain
sequence in TABLE 7. A conjugate or antibody construct can comprise a light
chain comprising
66
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
at least 80% sequence identity to a light chain sequence in TABLE 7. A
conjugate or antibody
construct can comprise an antibody heavy chain. A conjugate or antibody
construct can comprise
a heavy chain comprising a heavy chain sequence in TABLE 7. A conjugate or
antibody
construct can comprise a heavy chain comprising at least 80% sequence identity
to any heavy
chain sequence in TABLE 7 or TABLE 8. A conjugate or antibody construct can
comprise at
least 80% sequence identity to any sequence in TABLE 7. A conjugate or
antibody construct can
comprise a first binding domain and an Fc domain, wherein the first binding
domain and the Fc
domain comprise an antibody. A first binding domain can bind to a tumor
antigen. A conjugate
or antibody construct can comprise an antibody light chain. A conjugate or
antibody construct
can comprise a light chain comprising a light chain sequence in TABLE 7 or
TABLE 8. A
conjugate or antibody construct can comprise a light chain comprising at least
80% sequence
identity to a light chain sequence in TABLE 7 or TABLE 8. A conjugate or
antibody construct
can comprise an antibody heavy chain. A conjugate or antibody construct can
comprise a heavy
chain comprising a heavy chain sequence in TABLE 7 or TABLE 8. A conjugate or
antibody
construct can comprise a heavy chain comprising at least 80% sequence identity
to any heavy
chain sequence in TABLE 7 or TABLE 8. A conjugate or antibody construct can
comprise at
least 80% sequence identity to any sequence in TABLE 7 or TABLE 8.
[0192] A conjugate or antibody construct can comprise an anti-tumor antibody,
wherein the
antibody comprises: a) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 11, and a light chain sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 16; b) a heavy chain sequence
having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 24, and a light
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
29; c) a heavy
chain sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
37, and a light chain sequence having at least 80% sequence identity to an
amino acid sequence
of SEQ ID NO: 42; d) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 50, and a light chain sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 55; e) a heavy chain sequence
having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 63, and a light
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
68; f) a heavy
chain sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
76, and a light chain sequence having at least 80% sequence identity to an
amino acid sequence
of SEQ ID NO: 81; g) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 89, and a light chain sequence having at least 80%
sequence
67
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
identity to an amino acid sequence of SEQ ID NO: 94; h) a heavy chain sequence
having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 102, and a light
chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 107;
i) a heavy chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 115, and a light chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 120; j) a heavy chain sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 128, and a light chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 133; k) a
heavy chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 141,
and a light chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 146;1) a heavy chain sequence having at least 80% sequence identity
to an amino
acid sequence of SEQ ID NO: 154, and a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 159; m) a heavy chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 167, and a
light chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 172;
n) a heavy chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 180, and a light chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 185; o) a heavy chain sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 193, and a light chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 198; p) a
heavy chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 206,
and a light chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 211; q) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 813, and a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 814; r) a heavy chain
sequence having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 831, and a light
chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 832;
s) a heavy chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 219, and a light chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 224; t) a heavy chain sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 258, and a light chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 263; u) a
heavy chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 271,
and a light chain sequence having at least 80% sequence identity to an amino
acid sequence of
68
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
SEQ ID NO: 276; v) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 284, and a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 289; w) a heavy chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 297, and a
light chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 302;
x) a heavy chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 310, and a light chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 315; y) a heavy chain sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 323, and a light chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 328; z) a
heavy chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 336,
and a light chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 341; aa) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 349, and a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 354; bb) a heavy chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 362, and a
light chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 367;
cc) a heavy chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 375, and a light chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 380; dd) a heavy chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 388, and a light chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 393; ee) a
heavy chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 401,
and a light chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 406; ff) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 414, and a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 419; gg) a heavy chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 427, and a
light chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 432;
hh) a heavy chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 440, and a light chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 445; ii) a heavy chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 453, and a light chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 458; jj) a
heavy chain
69
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 466,
and a light chain sequence having at least 80% sequence identity to an amino
acid sequence of
SEQ ID NO: 471; kk) a heavy chain sequence having at least 80% sequence
identity to an amino
acid sequence of SEQ ID NO: 479, and a light chain sequence having at least
80% sequence
identity to an amino acid sequence of SEQ ID NO: 484; 11) a heavy chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 492, and a
light chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 497;
or mm) a heavy chain sequence having at least 80% sequence identity to an
amino acid sequence
of SEQ ID NO: 681, and a light chain sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 682.
[0193] A conjugate or antibody construct can comprise a second binding domain.
A conjugate or
antibody construct can comprise a second binding domain that specifically
binds to an antigen. A
conjugate or antibody construct can comprise a second binding domain that
specifically binds to
a molecule on an immune cell. An immune cell can be a T cell, B cell,
dendritic cell, macrophage,
NK cell, or NKT cell. In some embodiments, an immune cell is a T cell, B cell,
NK cell, or NKT
cell. In some embodiments, an immune cell is an antigen presenting cell. A
conjugate or
antibody construct can comprise a second binding domain that specifically
binds to a molecule
on an immune cell such as an antigen presenting cell. An antigen presenting
cell can be a
dendritic cell or a macrophage. A second binding domain can specifically bind
to a molecule on
an immune cell, wherein the molecule comprises at least 80% homology to an
amino acid
sequence of a group consisting of CD40, DEC-205, CD36 mannose scavenger
receptor 1,
CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A,
Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, CD32B, PD-L1,
and
CD47. A second binding domain can specifically bind to a molecule on an immune
cell, wherein
the molecule comprises at least 80% homology to an amino acid sequence of a
group consisting
of DEC-205, CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-
2,
OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64,
CD32A, CD16A, HVEM, and CD32B. A second binding domain can specifically bind
to a
molecule on an immune cell, wherein the molecule comprises at least 80%
homology to an
amino acid sequence of a group consisting of tumor necrosis factor receptor 2
(TNFR2) or
triggering receptor expressed on myeloid cells 2 (TREM2). A second binding
domain can
specifically bind to a molecule on an antigen presenting cell, wherein the
molecule comprises at
least 80% homology to a group consisting of CD40, DEC-205, CD36 mannose
scavenger
receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO,
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1,
CD47, and CD32B. A second binding domain can specifically bind to a molecule
on an antigen
presenting cell, wherein the molecule comprises at least 80% homology to a
group consisting of
DEC-205, CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2,
OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64,
CD32A, CD16A, HVEM, and CD32B. TABLE 2 shows exemplary amino acid sequences of
molecules on an immune cell to which a second binding domain can specifically
bind.
TABLE 2. Exemplary amino acid sequences of molecules on an immune cell to
which a second
binding domain can specifically bind
Immune Cell Molecule SEQ ID NO: Immune Cell Molecule SEQ ID NO:
CD40 (isoform I) 958 Dectin 1 (isoform 1) 997
CD40 (isoform II) 959 Dectin 1 (isoform 2) 998
DEC-205 (isoform 4) 960 Dectin 1 (isoform
3) 999
DEC-205 (isoform 2) 961 Dectin 1 (isoform
4) 1000
DEC-205 (isoform 5) 962 Dectin 1 (isoform
5) 1001
DEC-205 (isoform 3) 963 Dectin 1 (isoform
6) 1002
DEC-205 (isoform 1) 964 Dectin 1 (isoform
7) 1003
CD36 mannose Dectin 1 (isoform 8) 1004
scavenger receptor 1 965 Dectin 1 (isoform
9) 1005
(isoform 1) Dectin 1 (isoform 10) 1006
CD36 mannose Dectin 2 (isoform 1) 1007
scavenger receptor 1 966 Dectin 2 (isoform
2) 1008
(isoform 2) CLEC10A (isoform 1) 1009
CLEC9A 967 CLEC10A (isoform 2) 1010
DC-SIGN (isoform 1) 968 CLEC10A (isoform
3) 1011
DC-SIGN (isoform 2) 969 CD206 (isoform 1)
1012
DC-SIGN (isoform 3) 970 CD206 (isoform 2)
1013
DC-SIGN (isoform 4) 971 CD64 (isoform 1)
1014
DC-SIGN (isoform 5) 972 CD64 (isoform 2)
1015
DC-SIGN (isoform 6) 973 CD32A (isoform 1)
1016
DC-SIGN (isoform 7) 974 CD32A (isoform 2)
1017
DC-SIGN (isoform 8) 975 CD32A (isoform 3)
1018
DC-SIGN (isoform 9) 976 CD16A 1019
DC-SIGN (isoform 10) 977 HVEM (isoform 1) 1020
DC-SIGN (isoform 11) 978 HVEM (isoform 2) 1021
DC-SIGN (isoform 12) 979 CD32B (isoform IIB2) 1022
CLEC12A (isoform 2) 980 CD32B (isoform
IIB3) 1023
CLEC12A (isoform 1) 981 CD32B (isoform 4)
1024
CLEC12A (isoform 3) 982 CD32B (isoform 5)
1025
CLEC12A (isoform 4) 983 PD-Li (isoform 1)
955
CLEC12A (isoform 5) 984 PD-Li (isoform 2)
956
BDCA-2 (isoform 1) 985 PD-Li (isoform 3) 957
BDCA-2 (isoform 2) 986 CD47 (isoform 0A3-
OX4OL (isoform 1) 987 323) 1026
OX4OL (isoform 2) 988 CD47 (isoform 0A3-
1027
41BBL 989 293)
CD204 (isoform I) 990 CD47 (isoform 0A3-
1028
CD204 (isoform II) 991 305)
CD204 (isoform III) 992 CD47 (isoform 0A3-
1029
MARCO (isoform 1) 993 312)
MARCO (isoform 2) 994
CLEC5A (isoform 1) 995
CLEC5A (isoform 2) 996
71
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0194] In some embodiments, an amino acid sequence of the antigen on the
antigen presenting
cell has at least 80% sequence identity with the amino acid sequence of an
antigen selected from
the group consisting of CD40, DEC-205, CD36 mannose scavenger receptor 1,
CLEC9A, DC-
SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2,
CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B, and CD47, but not CD40
when the first binding domain specifically binds to HER2.
[0195] In some embodiments, an amino acid sequence of the antigen on the
antigen presenting
cell has at least 80% sequence identity with the amino acid sequence of an
antigen selected from
TABLE 2. In some embodiments, the second binding domain is a CD40 agonist. In
some
embodiments, the first binding domain comprises a single chain variable
fragment (scFv). In
some embodiments, the second binding domain is a single chain variable
fragment (scFv). In
some embodiments, the second binding domain comprises a single chain variable
fragment from
an anti-CD40 antibody, an anti-DEC-205 antibody, an anti-CD36 mannose
scavenger receptor 1
antibody, an anti-DC-SIGN antibody, an anti-CLEC9A antibody, an anti-CLEC12A
antibody, an
anti-BDCA-2 antibody, an anti-OX4OL antibody, an anti-41BBL antibody, an anti-
CD204
antibody, an anti-MARCO antibody, an anti-CLEC5A antibody, an anti-Dectin 1
antibody, an
anti-Dectin 2 antibody, an anti-CLEC10A antibody, an anti-CD206 antibody, an
anti-CD64
antibody, an anti-CD32A antibody, an anti-CD16A antibody, an anti-HVEM
antibody, an anti-
PD-L1, or an anti-CD32B antibody.
[0196] A conjugate or antibody construct can comprise an Fc domain. A
conjugate or antibody
construct can comprise a first binding domain, a second binding domain, and an
Fc domain,
wherein the first binding domain is attached to the Fc domain. A conjugate or
antibody construct
can comprise a first binding domain, a second binding domain, and an Fc
domain, wherein the
second binding domain is attached to the Fc domain. A first binding domain can
be attached to an
Fc domain as a fusion peptide. A second binding domain can be attached to an
Fc domain as a
fusion peptide. A first binding domain can be attached to an Fc domain via a
linker. A second
binding domain can be attached to an Fc domain via a linker.
[0197] A conjugate or antibody construct can comprise a second binding domain
comprising one
or more CDRs. A second binding domain can comprise a sequence or pair of
sequences in
TABLE 11. A second binding domain can comprise a sequence or pair of sequences
in TABLE
11 or TABLE 12.
[0198] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds CD40. A conjugate can comprise a second binding domain that is a CD40
agonist. A
72
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
conjugate or antibody construct can comprise a second binding domain that
binds CD40, wherein
the second binding domain comprises at least 80% sequence identity to: a)
HCDR1 comprising
an amino acid sequence of SEQ ID NO: 3, HCDR2 comprising an amino acid
sequence of SEQ
ID NO: 4, HCDR3 comprising an amino acid sequence of SEQ ID NO: 5, LCDR1
comprising an
amino acid sequence of SEQ ID NO: 8, LCDR2 comprising an amino acid sequence
of SEQ ID
NO: 9, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 10; b) HCDR1
comprising an amino acid sequence of SEQ ID NO: 582, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 583, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 584,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 587, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 588, and LCDR3 comprising an amino acid sequence
of SEQ ID
NO: 589; c) HCDR1 comprising an amino acid sequence of SEQ ID NO: 592, HCDR2
comprising an amino acid sequence of SEQ ID NO: 593, HCDR3 comprising an amino
acid
sequence of SEQ ID NO: 594, LCDR1 comprising an amino acid sequence of SEQ ID
NO: 597,
LCDR2 comprising an amino acid sequence of SEQ ID NO: 598, and LCDR3
comprising an
amino acid sequence of SEQ ID NO: 599; d) HCDR1 comprising an amino acid
sequence of
SEQ ID NO: 602, HCDR2 comprising an amino acid sequence of SEQ ID NO: 603,
HCDR3
comprising an amino acid sequence of SEQ ID NO: 604, LCDR1 comprising an amino
acid
sequence of SEQ ID NO: 607, LCDR2 comprising an amino acid sequence of SEQ ID
NO: 608,
and LCDR3 comprising an amino acid sequence of SEQ ID NO: 609; e) HCDR1
comprising an
amino acid sequence of SEQ ID NO: 612, HCDR2 comprising an amino acid sequence
of SEQ
ID NO: 613, HCDR3 comprising an amino acid sequence of SEQ ID NO: 614, LCDR1
comprising an amino acid sequence of SEQ ID NO: 617, LCDR2 comprising an amino
acid
sequence of SEQ ID NO: 618, and LCDR3 comprising an amino acid sequence of SEQ
ID NO:
619; f) HCDR1 comprising an amino acid sequence of SEQ ID NO: 622, HCDR2
comprising an
amino acid sequence of SEQ ID NO: 623, HCDR3 comprising an amino acid sequence
of SEQ
ID NO: 624, LCDR1 comprising an amino acid sequence of SEQ ID NO: 627, LCDR2
comprising an amino acid sequence of SEQ ID NO: 628, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 629; or g) HCDR1 comprising an amino acid sequence of
SEQ ID NO:
632, HCDR2 comprising an amino acid sequence of SEQ ID NO: 633, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 634, LCDR1 comprising an amino acid sequence
of SEQ
ID NO: 637, LCDR2 comprising an amino acid sequence of SEQ ID NO: 638, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 639.
[0199] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds DC-SIGN. A conjugate or antibody construct can comprise a second binding
domain that
73
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
binds DC-SIGN, wherein the second binding domain comprises at least 80%
sequence identity to:
a) HCDR1 comprising an amino acid sequence of SEQ ID NO: 640, HCDR2 comprising
an
amino acid sequence of SEQ ID NO: 641, HCDR3 comprising an amino acid sequence
of SEQ
ID NO: 642, LCDR1 comprising an amino acid sequence of SEQ ID NO: 643, LCDR2
comprising an amino acid sequence of SEQ ID NO: 644, and LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 645; b) HCDR1 comprising an amino acid sequence of SEQ
ID NO:
646, HCDR2 comprising an amino acid sequence of SEQ ID NO: 647, HCDR3
comprising an
amino acid sequence of SEQ ID NO: 648, LCDR1 comprising an amino acid sequence
of SEQ
ID NO: 649, LCDR2 comprising an amino acid sequence of SEQ ID NO: 650, and
LCDR3
comprising an amino acid sequence of SEQ ID NO: 651; or c) HCDR1 comprising an
amino acid
sequence of SEQ ID NO: 652, HCDR2 comprising an amino acid sequence of SEQ ID
NO: 653,
HCDR3 comprising an amino acid sequence of SEQ ID NO: 654, LCDR1 comprising an
amino
acid sequence of SEQ ID NO: 655, LCDR2 comprising an amino acid sequence of
SEQ ID NO:
656, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 657.
[0200] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds DEC-205. A conjugate or antibody construct comprising a second binding
domain that
binds DEC-205 can comprise at least 80% sequence identity to: a) HCDR1
comprising an amino
acid sequence of SEQ ID NO: 234, HCDR2 comprising an amino acid sequence of
SEQ ID NO:
235, HCDR3 comprising an amino acid sequence of SEQ ID NO: 236, LCDR1
comprising an
amino acid sequence of SEQ ID NO: 239, LCDR2 comprising an amino acid sequence
of SEQ
ID NO: 240, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 241;
orb) HCDR1
comprising an amino acid sequence of SEQ ID NO: 247, HCDR2 comprising an amino
acid
sequence of SEQ ID NO: 248, HCDR3 comprising an amino acid sequence of SEQ ID
NO: 249,
LCDR1 comprising an amino acid sequence of SEQ ID NO: 252, LCDR2 comprising an
amino
acid sequence of SEQ ID NO: 253, and LCDR3 comprising an amino acid sequence
of SEQ ID
NO: 254.
[0201] A conjugate or antibody construct can comprise a second binding domain
comprising one
or more variable domains. A conjugate or antibody construct can comprise a
second binding
domain comprising a light chain variable domain (VL domain). A second binding
domain can
comprise at least 80% sequence identity to any VL sequence in TABLE 13. A
conjugate or
antibody construct can comprise a second binding domain comprising a heavy
chain variable
domain. A second binding domain can comprise at least 80% sequence identity to
any VH
sequence in TABLE 13. A second binding domain can comprise at least 80%
sequence identity
to any sequence in TABLE 13. A second binding domain can comprise at least 80%
sequence
74
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
identity to any VL sequence in TABLE 13 or TABLE 14. A conjugate or antibody
construct can
comprise a second binding domain comprising a heavy chain variable domain. A
second binding
domain can comprise at least 80% sequence identity to any VH sequence in TABLE
13 or
TABLE 14. A second binding domain can comprise at least 80% sequence identity
to any
sequence in TABLE 13 or TABLE 14.
[0202] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds CD40. A conjugate or antibody construct can comprise a second binding
domain that is a
CD40 agonist. A conjugate or antibody construct can comprise a second binding
domain that
binds CD40, wherein the second binding domain comprises: a) a VH sequence
having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 2, and a VL
sequence having
at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 7; b) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
581, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 586; c)
a VH sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
591, and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ
ID NO: 596; d) a VH sequence having at least 80% sequence identity to an amino
acid sequence
of SEQ ID NO: 601, and a VL sequence having at least 80% sequence identity to
an amino acid
sequence of SEQ ID NO: 606; e) a VH sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 611, and a VL sequence having at least 80%
sequence
identity to an amino acid sequence of SEQ ID NO: 616; f) a VH sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 621, and a VL
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 626; g) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
631, and a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 636.
[0203] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds DEC-205. A conjugate or antibody construct can comprise a second binding
domain that
binds DEC-205, wherein the second binding domain comprises: a) a VH sequence
having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 233, and a VL
sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
238; or b) a VH
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 246,
and a VL sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 251.
[0204] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds CD36 mannose scavenger receptor 1. A conjugate or antibody construct can
comprise a
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
second binding domain that binds CD36 mannose scavenger receptor 1, wherein
the second
binding domain comprises a VH sequence having at least 80% sequence identity
to an amino
acid sequence of SEQ ID NO: 658, and a VL sequence having at least 80%
sequence identity to
an amino acid sequence of SEQ ID NO: 659.
[0205] A conjugate or antibody construct can comprise a second binding domain
that specifically
binds CLEC9A. A conjugate or antibody construct can comprise a second binding
domain that
binds CLEC9A, wherein the second binding domain comprises a VH sequence having
at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 660, and a VL
sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
661.
[0206] A conjugate or antibody construct can comprise a second binding domain
and an Fc
domain, wherein the second binding domain and the Fc domain comprise an
antibody. A
conjugate or antibody construct can comprise a heavy chain and a light chain
that target a
molecule expressed by an immune cell such as an antigen presenting cell. A
conjugate or
antibody construct can comprise an antibody light chain. A conjugate or
antibody construct can
comprise a light chain comprising at least 80% sequence identity to any light
chain sequence in
TABLE 15. A conjugate or antibody construct can comprise an antibody heavy
chain. A
conjugate or antibody construct can comprise a heavy chain comprising at least
80% sequence
identity to any heavy chain sequence in TABLE 15. A conjugate or antibody
construct can
comprise at least 80% sequence identity to any sequence in TABLE 15. A
conjugate or antibody
construct can comprise a light chain comprising at least 80% sequence identity
to any light chain
sequence in TABLE 15 or TABLE 16. A conjugate or antibody construct can
comprise an
antibody heavy chain. A conjugate or antibody construct can comprise a heavy
chain comprising
at least 80% sequence identity to any heavy chain sequence in TABLE 15 or
TABLE 16. A
conjugate or antibody construct can comprise at least 80% sequence identity to
any sequence in
TABLE 15 or TABLE 16.
[0207] A conjugate or antibody construct can comprise a heavy chain and a
light chain that target
a molecule expressed by an immune cell such as an antigen presenting cell. A
conjugate or
antibody construct can comprise a first binding domain and an Fc domain,
wherein the first
binding domain and the Fc domain comprise an antibody. A conjugate or antibody
construct can
comprise an anti-CD40 antibody, the conjugate comprising: a) a heavy chain
sequence having at
least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1 and a
light chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 6; b) a
heavy chain sequence having at least 80% sequence identity to an amino acid
sequence of SEQ
ID NO: 577 or SEQ ID NO: 578, and a light chain sequence having at least 80%
sequence
76
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
identity to an amino acid sequence of SEQ ID NO: 579; c) a heavy chain
sequence having at least
80% sequence identity to an amino acid sequence of SEQ ID NO: 580, and a light
chain
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 585; d)
a heavy chain sequence having at least 80% sequence identity to an amino acid
sequence of SEQ
ID NO: 590, and a light chain sequence having at least 80% sequence identity
to an amino acid
sequence of SEQ ID NO: 595; e) a heavy chain sequence having at least 80%
sequence identity
to an amino acid sequence of SEQ ID NO: 600, and a light chain sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 605; f) a heavy
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
610, and a light
chain sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
615; g) a heavy chain sequence having at least 80% sequence identity to an
amino acid sequence
of SEQ ID NO: 620, and a light chain sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 625; or h) a heavy chain sequence having at
least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 630, and a light
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
635.
[0208] A conjugate or antibody construct can comprise a first binding domain
and an Fc domain,
wherein the first binding domain and the Fc domain comprise an antibody. A
conjugate or
antibody construct can comprise an anti-DEC-205 antibody, the conjugate or
antibody construct
comprising: a) a heavy chain sequence having at least 80% sequence identity to
an amino acid
sequence of SEQ ID NO: 232, and a light chain sequence having at least 80%
sequence identity
to an amino acid sequence of SEQ ID NO: 237; orb) a heavy chain sequence
having at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 245, and a light
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
250.
[0209] A conjugate or antibody construct can comprise a first binding domain
and an Fc domain,
wherein the first binding domain and the Fc domain comprise an antibody. A
conjugate or
antibody construct can comprise an anti-CLEC12A antibody, the conjugate
comprising: a) a
heavy chain sequence having at least 80% sequence identity to an amino acid
sequence of SEQ
ID NO: 662, and a light chain sequence having at least 80% sequence identity
to an amino acid
sequence of SEQ ID NO: 665; b) a heavy chain sequence having at least 80%
sequence identity
to an amino acid sequence of SEQ ID NO: 663, and a light chain sequence having
at least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 665; or c) a heavy
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
664, and a light
chain sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
665.
77
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0210] A conjugate or antibody construct can comprise a first binding domain
and an Fc domain,
wherein the first binding domain and the Fc domain comprise an antibody. A
conjugate or
antibody construct can comprise an anti-BDCA-2 antibody, the conjugate
comprising: a) a heavy
chain sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
666, and a light chain sequence having at least 80% sequence identity to an
amino acid sequence
of SEQ ID NO: 669; b) a heavy chain sequence having at least 80% sequence
identity to an
amino acid sequence of SEQ ID NO: 667, and a light chain sequence having at
least 80%
sequence identity to an amino acid sequence of SEQ ID NO: 670; or c) a heavy
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
668, and a light
chain sequence having at least 80% sequence identity to an amino acid sequence
of SEQ ID NO:
671.
Antibody-ScFv Fusion Protein Products
[0211] A conjugate or antibody construct can comprise a first binding domain,
a second binding
domain, and an Fc domain, wherein the first binding domain and the second
binding domain are
attached to the Fc domain. The first binding domain and the second binding
domain can be
attached to the Fc domain as a fusion peptide (also referred to as a fusion
protein). The first
binding domain can be attached to the Fc domain at an N-terminal end of the Fc
domain, wherein
the second binding domain can be attached to the Fc domain at a C-terminal
end. The first
binding domain can be attached to the Fc domain at an N-terminal end of the Fc
domain, wherein
the second binding domain can be attached to the Fc domain at a C-terminal end
via a
polypeptide linker ranging from 10 to 25 amino acids comprising the sequence
[G45]n where n =
2 to 5 (SEQ ID NO: 1330). Alternatively, the first binding domain can be
attached to the Fc
domain at a C-terminal end of the Fc domain, wherein the second binding domain
can be
attached to the Fc domain at an N-terminal end. A second binding domain and an
Fc domain can
comprise an antibody and a first binding domain can comprise a single chain
variable fragment
(scFv). A single chain variable fragment can comprise a heavy chain variable
domain and a light
chain variable domain of an antibody. The first binding domain of the fusion
peptide can be
attached to the second binding domain at a heavy chain variable domain of the
single chain
variable fragment of the first binding domain (HL orientation). Alternatively,
the first binding
domain of the fusion peptide can be attached to the second binding domain at a
light chain
variable domain of the single chain variable fragment of the first binding
domain (LH
orientation). In either orientation, the first binding domain and the second
binding domain can be
attached via a polypeptide linker varying in length from 15 to 25 amino acids,
wherein the linker
comprises the sequence [G45]n where n = 3 to 5 (SEQ ID NO: 1331).
78
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0212] Alternatively, a first binding domain and an Fc domain can comprise an
antibody and the
second binding domain can comprise a single chain variable fragment (scFv).
The second
binding domain of the fusion peptide can be attached to the first binding
domain at a heavy chain
variable domain of the single chain variable fragment of the first binding
domain (HL
orientation). Alternatively, the second binding domain of the fusion peptide
can be attached to
the first binding domain at a light chain variable domain of the single chain
variable fragment of
the first binding domain (LH orientation).
[0213] A conjugate or antibody construct can comprise a first binding domain
and a second
binding domain, wherein the second binding domain can be attached to the first
binding domain.
The conjugate or antibody construct can comprise an antibody comprising a
light chain and a
heavy chain. The first binding domain can comprise a Fab fragment of the light
and heavy chains.
The second binding domain can be attached to the light chain at a C-terminus
or C-terminal end
of the light chain as a fusion peptide. The second binding domain can comprise
a single chain
variable fragment (scFv).
[0214] A conjugate or antibody construct can comprise a first binding domain,
a second binding
domain, and an Fc domain, wherein the first binding domain and the second
binding domain are
attached to the Fc domain as a fusion peptide. The second binding domain of
the fusion peptide
can specifically bind to an antigen with at least 80% homology to CD40. The
second binding
domain of the fusion peptide can be a CD40 agonist. The first binding domain
of the fusion
peptide can target a tumor antigen. The conjugate or antibody construct can
comprise a fusion
peptide comprising a heavy chain (HC) attached to a single chain variable
fragment. The
conjugate or antibody construct comprising the fusion peptide can comprise a
sequence in
TABLE 9. The conjugate or antibody construct comprising the fusion peptide can
comprise at
least 80% sequence identity to any sequence in TABLE 9. The conjugate or
antibody construct
comprising the fusion peptide can comprise at least 80% sequence identity to a
sequence of a
heavy chain CD40 monoclonal antibody (mAb) with tumor ScFv in TABLE 9. The
conjugate or
antibody construct can comprise a fusion peptide comprising a sequence of a
heavy chain CD40
mAb with tumor ScFv in TABLE 9 and a light chain comprising SEQ ID NO: 6. The
conjugate
or antibody construct can comprise a fusion peptide comprising at least 80%
sequence identity to
a sequence of a heavy chain CD40 mAb with tumor ScFv in TABLE 9 and a light
chain
comprising at least 80% sequence identity to SEQ ID NO: 6. The conjugate or
antibody construct
comprising the fusion peptide can comprise a sequence of a heavy chain tumor
mAb with CD40
ScFv in TABLE 9. The conjugate or construct comprising the fusion peptide can
comprise at
least 80% sequence identity to any sequence of a heavy chain tumor mAb with
CD40 ScFv in
79
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
TABLE 9. The conjugate or antibody construct comprising the fusion peptide can
comprise a
sequence of a heavy chain tumor antigen mAb with CD40 ScFv in TABLE 9, and a
light chain
mAb for the tumor antigen in TABLE 7. The conjugate or antibody construct
comprising the
fusion peptide can comprise at least 80% sequence identity to any sequence of
a heavy chain
tumor antigen mAb with CD40 ScFv in TABLE 9, and at least 80% sequence
identity to a light
chain mAb for the tumor antigen in TABLE 7.
[0215] The conjugate or antibody construct comprising the fusion peptide can
comprise a
sequence in TABLE 9 or TABLE 10. The conjugate or antibody construct
comprising the fusion
peptide can comprise at least 80% sequence identity to any sequence in TABLE 9
or TABLE 10.
The conjugate or antibody construct comprising the fusion peptide can comprise
at least 80%
sequence identity to a sequence of a heavy chain CD40 monoclonal antibody
(mAb) with tumor
ScFv in TABLE 9 or TABLE 10. The conjugate or antibody construct can comprise
a fusion
peptide comprising a sequence of a heavy chain CD40 mAb with tumor ScFv in
TABLE 9 or
TABLE 10 and a light chain comprising SEQ ID NO: 6. The conjugate or antibody
construct can
comprise a fusion peptide comprising at least 80% sequence identity to a
sequence of a heavy
chain CD40 mAb with tumor ScFv in TABLE 9 or TABLE 10 and a light chain
comprising at
least 80% sequence identity to SEQ ID NO: 6. The conjugate or antibody
construct comprising
the fusion peptide can comprise a sequence of a heavy chain tumor mAb with
CD40 ScFv in
TABLE 9 or TABLE 10. The conjugate or construct comprising the fusion peptide
can comprise
at least 80% sequence identity to any sequence of a heavy chain tumor mAb with
CD40 ScFv in
TABLE 9 or TABLE 10. The conjugate or antibody construct comprising the fusion
peptide can
comprise a sequence of a heavy chain tumor antigen mAb with CD40 ScFv in TABLE
9 or
TABLE 10, and a light chain mAb for the tumor antigen in TABLE 7 or TABLE 8.
The
conjugate or antibody construct comprising the fusion peptide can comprise at
least 80%
sequence identity to any sequence of a heavy chain tumor antigen mAb with CD40
ScFv in
TABLE 9 or TABLE 10, and at least 80% sequence identity to a light chain mAb
for the tumor
antigen in TABLE 7 or TABLE 8.
[0216] A conjugate or antibody construct can comprise a first binding domain
and a second
binding domain, wherein the second binding domain can be attached to the first
binding domain.
A conjugate or antibody construct can comprise a first binding domain, a
second binding domain,
and an Fc domain, wherein the second binding domain can be attached to the
first binding
domain. The second binding domain can be attached at a C-terminal end of the
first binding
domain as a fusion peptide. The first binding domain can comprise a Fab
fragment comprising a
light chain, wherein the second binding domain can be attached at a C-terminal
end of the light
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
chain as a fusion peptide. The second binding domain of the fusion peptide can
comprise a single
chain variable fragment (scFv). The second binding domain of the fusion
peptide can be attached
to the first binding domain at a heavy chain variable domain of the single
chain variable fragment
of the first binding domain (HL orientation). Alternatively, the second
binding domain of the
fusion peptide can be attached to the first binding domain at a light chain
variable domain of the
single chain variable fragment of the first binding domain (LH orientation).
For example, a
fusion peptide comprising a light chain of an anti-CEA antibody attached to an
anti-CD40 scFv
in the LH orientation can be illustrated by SEQ ID NO: 842. All fusion
sequences comprising an
scFv sequence are in the HL orientation unless indicated otherwise (e.g.,
sequence name recites
"(LH)" indicating light heavy orientation).
[0217] The first binding domain of the fusion peptide can target a tumor
antigen. The second
binding domain of the fusion peptide can target an APC antigen. The second
binding domain of
the fusion peptide can target CD40. The first binding domain can comprise a
Fab fragment
comprising a light chain, wherein the second binding domain is attached at a C-
terminal end of
the light chain as a fusion peptide. The conjugate or antibody construct
comprising the fusion
peptide can comprise a sequence in TABLE 18. The conjugate or antibody
construct comprising
the fusion peptide can comprise at least 80% sequence identity to any sequence
in TABLE 18.
The conjugate or antibody construct comprising the fusion peptide can comprise
a sequence of a
light chain CD40 mAb with tumor ScFv in TABLE 18. The conjugate or antibody
construct
comprising the fusion peptide can comprise at least 80% sequence identity to
any sequence of a
light chain CD40 mAb with tumor ScFv in TABLE 18. The conjugate or antibody
construct can
comprise a fusion peptide comprising a sequence of a light chain CD40 mAb with
tumor ScFv in
TABLE 11 and a heavy chain comprising SEQ ID NO: 1. The conjugate or antibody
construct
can comprise a fusion peptide comprising at least 80% sequence identity to any
sequence of a
light chain CD40 mAb with tumor ScFv in TABLE 11 and a heavy chain comprising
at least
80% sequence identity to SEQ ID NO: 1. The conjugate or antibody construct
comprising the
fusion peptide can comprise a sequence of a light chain tumor mAb with CD40
ScFv in TABLE
18. The conjugate or antibody construct comprising the fusion peptide can
comprise at least 80%
sequence identity to any sequence of a light chain tumor mAb with CD40 ScFv in
TABLE 18.
The conjugate or antibody construct can comprise a fusion peptide comprising a
sequence of a
light chain tumor antigen mAb with CD40 ScFv in TABLE 18, and a heavy chain
mAb for the
tumor antigen in TABLE 7. The conjugate or antibody construct can comprise a
fusion peptide
comprising at least 80% sequence identity to any sequence of a light chain
tumor antigen mAb
with CD40 ScFv in TABLE 18, and at least 80% sequence identity to a heavy
chain mAb for the
81
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
tumor antigen in TABLE 7. The conjugate or antibody construct can comprise a
fusion peptide
comprising a sequence of a light chain tumor antigen mAb with CD40 ScFv in
TABLE 18, and a
heavy chain mAb for the tumor antigen in TABLE 7 or TABLE 8. The conjugate or
antibody
construct can comprise a fusion peptide comprising at least 80% sequence
identity to any
sequence of a light chain tumor antigen mAb with CD40 ScFv in TABLE 18, and at
least 80%
sequence identity to a heavy chain mAb for the tumor antigen in TABLE 7 or
TABLE 8.
[0218] A conjugate or antibody construct can comprise a first binding domain,
a second binding
domain, and an Fc domain, wherein the first binding domain and the second
binding domain are
attached to the Fc domain as a fusion peptide. The first binding domain of the
fusion peptide can
specifically bind to an antigen with at least 80% homology to DEC-205. The
second binding
domain of the fusion peptide can target a tumor antigen. The conjugate or
antibody construct can
comprise a fusion peptide comprising a heavy chain attached to a single chain
variable fragment.
The conjugate or antibody construct comprising the fusion peptide can comprise
a sequence in
TABLE 17. The conjugate or antibody construct comprising the fusion peptide
can comprise at
least 80% sequence identity to any sequence in TABLE 17. The conjugate or
antibody construct
comprising the fusion peptide can comprise a sequence of a heavy chain DEC-205
mAb with
tumor ScFv in TABLE 17. The conjugate or antibody construct comprising the
fusion peptide
can comprise at least 80% sequence identity to any sequence of a heavy chain
DEC-205 mAb
with tumor ScFv in TABLE 17. The conjugate or antibody construct can comprise
a fusion
peptide comprising a sequence of a heavy chain DEC-205 mAb with tumor ScFv in
TABLE 10
and a peptide comprising SEQ ID NO: 237. The conjugate or antibody construct
can comprise a
fusion peptide comprising at least 80% sequence identity to any sequence of a
heavy chain DEC-
205 mAb with tumor ScFv in TABLE 17 and a peptide comprising at least 80%
sequence
identity to SEQ ID NO: 237. The conjugate or antibody construct comprising the
fusion peptide
can comprise a sequence of a heavy chain tumor antigen mAb with CD40 ScFv in
TABLE 17.
The conjugate or antibody construct comprising the fusion peptide can comprise
at least 80%
sequence identity to any sequence of a heavy chain tumor antigen mAb with CD40
ScFv in
TABLE 17. The conjugate or antibody construct comprising the fusion peptide
can comprise a
sequence of a heavy chain tumor antigen mAb with CD40 ScFv in TABLE 17, and a
heavy
chain mAb for the tumor antigen in TABLE 7. The conjugate or antibody
construct comprising
the fusion peptide can comprise at least 80% sequence identity to any sequence
of a heavy chain
tumor antigen mAb with CD40 ScFv in TABLE 17, and at least 80% sequence
identity to a
heavy chain mAb for the tumor antigen in TABLE 7. The conjugate or antibody
construct
comprising the fusion peptide can comprise a sequence of a heavy chain tumor
antigen mAb with
82
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
CD40 ScFv in TABLE 17, and a heavy chain mAb for the tumor antigen in TABLE 7
or
TABLE 8. The conjugate or antibody construct comprising the fusion peptide can
comprise at
least 80% sequence identity to any sequence of a heavy chain tumor antigen mAb
with CD40
ScFv in TABLE 17, and at least 80% sequence identity to a heavy chain mAb for
the tumor
antigen in TABLE 7 or TABLE 8.
[0219] The second binding domain of the fusion peptide can target DEC-205. The
conjugate or
antibody construct comprising the fusion peptide can comprise a sequence in
TABLE 19. The
conjugate or antibody construct comprising the fusion peptide can comprise at
least 80%
sequence identity to any sequence in TABLE 19. The conjugate or antibody
construct
comprising the fusion peptide can comprise a sequence of a light chain DEC-205
mAb with
tumor ScFv in TABLE 19. The conjugate or antibody construct comprising the
fusion peptide
can comprise at least 80% sequence identity to any sequence of a light chain
DEC-205 mAb with
tumor ScFv in TABLE 19. The conjugate or antibody construct comprising a
fusion peptide can
comprise a sequence of a light chain DEC-205 mAb with tumor ScFv in TABLE 19
and SEQ ID
NO: 237. The conjugate or antibody construct comprising a fusion peptide can
comprise at least
80% sequence identity to any sequence of a light chain DEC-205 mAb with tumor
ScFv in
TABLE 19 and at least 80% sequence identity to SEQ ID NO: 237. The conjugate
or antibody
construct comprising the fusion peptide can comprise a sequence of a light
chain tumor mAb
with DEC-205 ScFv in TABLE 19. The conjugate or antibody construct comprising
the fusion
peptide can comprise at least 80% sequence identity to any sequence of a light
chain tumor mAb
with DEC-205 ScFv in TABLE 19. The conjugate or antibody construct comprising
a fusion
peptide can comprise a sequence of a light chain tumor antigen mAb with DEC-
205 ScFv in
TABLE 19, and a heavy chain mAb for the tumor antigen in TABLE 7. The
conjugate or
antibody construct comprising a fusion peptide can comprise at least 80%
sequence identity to
any sequence of a light chain tumor antigen mAb with DEC-205 ScFv in TABLE 19,
and at least
80% sequence identity to a heavy chain mAb for the tumor antigen in TABLE 7.
The conjugate
or antibody construct comprising a fusion peptide can comprise a sequence of a
light chain tumor
antigen mAb with DEC-205 ScFv in TABLE 19, and a heavy chain mAb for the tumor
antigen
in TABLE 7 or TABLE 8. The conjugate or antibody construct comprising a fusion
peptide can
comprise at least 80% sequence identity to any sequence of a light chain tumor
antigen mAb with
DEC-205 ScFv in TABLE 19, and at least 80% sequence identity to a heavy chain
mAb for the
tumor antigen in TABLE 7 or TABLE 8.
[0220] The second binding domain of the fusion peptide can specifically bind
to an antigen of an
immune cell, such as an antigen presenting cell, (APC). The second binding
domain of the fusion
83
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
peptide can specifically bind to an antigen with at least 80% homology to
CD40. The second
binding domain of the fusion peptide can be a CD40 agonist. The second binding
domain of the
fusion peptide can specifically bind to an antigen with at least 80% homology
to DEC-205. The
second binding domain of the fusion peptide can specifically bind to an
antigen with at least 80%
homology to DC-SIGN. The second binding domain of the fusion peptide can
specifically bind to
an antigen with at least 80% homology to CD36 mannose scavenger receptor. The
second
binding domain of the fusion peptide can specifically bind to an antigen with
at least 80%
homology to CLEC12A. The second binding domain of the fusion peptide can
specifically bind
to an antigen with at least 80% homology to BDCA-2. The second binding domain
of the fusion
peptide can specifically bind to an antigen having an amino acid sequence with
at least 80% or
100% homology to an amino acid sequence of CD40, CD47, DEC-205, CD36 mannose
scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204,
MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM,
PD-L1, or CD32BThe second binding domain of the fusion peptide can
specifically bind to an
antigen having an amino acid sequence with at least 80% or 100% homology to an
amino acid
sequence of CD40, CD47, DEC-205, CD36 mannose scavenger receptor 1, CLEC9A, DC-
SIGN,
CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2,
CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B, TNFR2, or TREM2. The
second binding domain of the fusion peptide can specifically bind to an
antigen having an amino
acid sequence with at least 80% or 100% homology to an amino acid sequence of
CD40, DEC-
205, CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2,
OX4OL,
41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A,
CD16A, HVEM, PD-L1, or CD32B. The second binding domain of the fusion peptide
can
specifically bind to an antigen having an amino acid sequence with at least
80% or 100%
homology to an amino acid sequence of CD40, DEC-205, CD36 mannose scavenger
receptor 1,
CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A,
Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B,
TNFR2, or TREM2. The first binding domain of the fusion peptide can target a
tumor antigen.
The first binding domain of the fusion peptide can target an antigen having an
amino acid
sequence with at least 80% or 100% homology to an amino acid sequence of HER2,
EGFR,
CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2,
EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3,
or
NY-ESO-1. The first binding domain of the fusion peptide also can target an
antigen having an
amino acid sequence with at least 80% or 100% homology to the amino acid
sequence of HER2,
84
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA,
CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1,
MAGE-A3, NY-ESO-1, LRRC15, GLP-3, CLDN6, CLDN16, UPK1B, VTCN1 (B7-H4), or
STRA6. In some embodiments, the first targeting domain can target an antigen
having an amino
acid sequence with at least 80% or 100% homology to the amino acid sequence of
TROP2, CEA,
MUC16, LRRC15, CLDN6, CLDN16, UPK1B, VTCN1 (B7-H4) or STRA6.
[0221] Alternatively, the second binding domain of the fusion peptide can
target a tumor antigen.
The second binding domain of the fusion peptide can target an antigen having
an amino acid
sequence with at least 80% or 100% homology to the amino acid sequence of
HER2, EGFR,
CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2,
EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3,
or
NY-ESO-1. The second binding domain of the fusion peptide also can target an
antigen having
an amino acid sequence with at least 80% or 100% homology to the amino acid
sequence of
HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA,
CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1,
MAGE-A3, NY-ESO-1, LRRC15, GLP-3, CLDN6, CLDN16, UPK1B, VTCN1 (B7-H4), or
STRA6. The first binding domain of the fusion peptide can specifically bind to
an antigen having
an amino acid sequence with at least 80% homology to the amino acid sequence
of CD40. The
first binding domain of the fusion peptide can be a CD40 agonist. The first
binding domain of the
fusion peptide can specifically bind to an antigen having an amino acid
sequence with at least
80% homology to the amino acid sequence of DEC-205. The first binding domain
of the fusion
peptide can specifically bind to an antigen having an amino acid sequence with
at least 80% or
100% homology to the amino acid sequence of CD40, DEC-205, CD36 mannose
scavenger
receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO,
CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1,
or
CD32B. The first binding domain of the fusion peptide can specifically bind to
an antigen having
an amino acid sequence with at least 80% homology to the amino acid sequence
of DEC-205.
The first binding domain of the fusion peptide can specifically bind to an
antigen having an
amino acid sequence with at least 80% or 100% homology to the amino acid
sequence of CD40,
DEC-205, CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2,
OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64,
CD32A, CD16A, HVEM, PD-L1, CD32B, TNFR2, or TREM2.
[0222] In some embodiments, the first binding domain can specifically bind to
an antigen having
an amino acid sequence with at least 80% or 100% homology to the amino acid
sequence of
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
TROP2, CEA, MUC16, LRRC15, CLDN6, CLDN16, UPK1B, VTCN1 (B7-H4) and STRA6 and
a second binding domain can target an antigen having an amino acid sequence
with at least 80%
or 100% homology to the amino acid sequence of CD40 or PD-Li.
[0223] A conjugate or antibody construct can comprise a first binding domain,
a second binding
domain, and a third binding domain. A conjugate or antibody construct can
comprise a first
binding domain, a second binding domain, a third binding domain, and an Fc
domain. The first
binding domain and the second binding domain can be attached to the Fc domain.
The first and
second binding domains are described herein throughout the specification. The
first binding
domain can be attached to the Fc domain at an N-terminal end of the Fc domain.
The second
binding domain can be attached at a C-terminal end of the Fc domain. The
second binding
domain can be attached at a C-terminal end of the Fc domain via a polypeptide
linker having a
length of 10 to 25 amino acid comprising the sequence [G4S]n where n = 2 to 5
(SEQ ID NO:
1330). The third binding domain can be attached to a C-terminal end of the
first binding domain.
The third binding domain can be attached at a C-terminal end of the Fc domain
via a polypeptide
linker having a length of 10 to 25 amino acid comprising the sequence [G45]n
where n = 2 to 5
(SEQ ID NO: 1330). The third binding domain can be attached to a C-terminal
end of a light
chain of the first binding domain. One or more of the first binding domain,
the second binding
domain, the third binding domain, and the Fc domain can be attached as a
fusion peptide. The
first binding domain can comprise a Fab fragment comprising a light chain,
wherein the second
binding domain is attached at a C-terminal end of the light chain as a fusion
peptide. The second
binding domain of the fusion peptide can comprise a single chain variable
fragment (scFv). The
second binding domain of the fusion peptide can be attached to the Fc domain
at a heavy chain
variable domain of the single chain variable fragment of the second binding
domain (HL
orientation). The second binding domain of the fusion peptide can be attached
to the Fc domain
at a light chain variable domain of the single chain variable fragment of the
second binding
domain (LH orientation). The third binding domain of the fusion peptide can
comprise a single
chain variable fragment (scFv). The conjugate or antibody construct can
comprise a fusion
peptide comprising the third binding domain attached to the first binding
domain having at least
80% or 100% sequence identity to any sequence in TABLE 18 or TABLE 19. The
third binding
domain of the fusion peptide can be attached to the first binding domain at a
heavy chain variable
domain of the single chain variable fragment of the first binding domain (HL
orientation).
Alternatively, the third binding domain of the fusion peptide can be attached
to the first binding
domain at a light chain variable domain of the single chain variable fragment
of the first binding
domain (LH orientation). The third binding domain of the fusion peptide can
target an antigen of
86
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
an immune cell, such as an antigen presenting cell, (APC). The third binding
domain of the
fusion peptide can specifically bind to an antigen with at least 80% homology
to the amino acid
sequence of CD40. The third binding domain of the fusion peptide can be a CD40
agonist. The
third binding domain of the fusion peptide can specifically bind to an antigen
with at least 80%
homology to the amino acid sequence of DEC-205. The third binding domain of
the fusion
peptide can specifically bind to an antigen having an amino acid sequence with
at least 80%
homology to the amino acid sequence of CD40, DEC-205, CD36 mannose scavenger
receptor 1,
CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A,
Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, or CD32B.
The
third binding domain of the fusion peptide can specifically bind to an antigen
having an amino
acid sequence with at least 80% homology to the amino acid sequence of CD40,
DEC-205, CD36
mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL, 41BBL,
CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A,
HVEM, PD-L1, CD32B, TNFR2, or TREM2.
[0224] Alternatively, the third binding domain can target a tumor antigen. The
third binding
domain of the fusion peptide can target an antigen having an amino acid
sequence with at least
80% or 100% homology to the amino acid sequence of HER2, EGFR, CMET, HER3,
MUC1,
MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2,
RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, or NY-ESO-1. The third
binding domain of the fusion peptide can target an antigen having an amino
acid sequence with at
least 80% or 100% homology to the amino acid sequence of HER2, EGFR, CMET,
HER3,
MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP,
EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1,
LRRC15, GLP-3, CLDN6, CLDN16, UPK1B, VTCN1 (B7-H4), or STRA6.
[0225] A conjugate or antibody construct can comprise a first binding domain
targeting CD40
and a second binding domain targeting DEC-205. Alternatively, a conjugate or
antibody
construct can comprise a first binding domain targeting DEC-205 and a second
binding domain
targeting CD40. A conjugate or antibody construct can comprise a first binding
domain, a second
binding domain, and an Fc domain. The first binding domain and the second
binding domain can
be attached to the Fc domain. The first binding domain can be attached to the
Fc domain at an N-
terminal end of the Fc domain, wherein the second binding domain is attached
to the Fc domain
at a C-terminal end of the Fc domain. Alternatively, second binding domain can
be attached to
the Fc domain at an N-terminal end of the Fc domain, wherein the first binding
domain is
attached to the Fc domain at a C-terminal end of the Fc domain. A conjugate or
antibody
87
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
construct can comprise a fusion peptide comprising a first binding domain
targeting CD40 and a
second binding domain targeting DEC-205. The fusion peptide can comprise at
least 80% or
100% sequence identity to any sequence in TABLE 20.
[0226] Additionally, conjugates or antibody constructs containing the
sequences referenced in
TABLES 3-20 can have a dissociation constant (Kd) that is less than lOnM for
the antigen of the
first binding domain. The conjugates or antibody constructs containing the
sequences referenced
in TABLES 3-20 can have a dissociation constant (Kd) that is less than lOnM
for the antigen of
the second binding domain. The conjugates or antibody constructs containing
the sequences
referenced in TABLES 3-20 can have a dissociation constant (Kd) that is less
than 10 nM for the
antigen of the third binding domain. The conjugates or antibody constructs can
have a
dissociation constant (Kd) for the antigen of the first binding domain that is
less than 1 nM, less
than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. The
conjugates or antibody
constructs can have a dissociation constant (Kd) for the antigen of the second
binding domain
that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or
less than 0.1 pM.
The conjugates or antibody constructs can have a dissociation constant (Kd)
for the antigen of the
third binding domain that is less than 1 nM, less than 100 pM, less than 10
pM, less than 1 pM,
or less than 0.1 pM.
[0227] An anti-CD40 light chain can be expressed with its corresponding anti-
CD40 heavy chain
or fragment thereof. The corresponding anti-CD40 heavy chain or fragment
thereof can be a
heavy chain or fragment that when paired with the anti-CD40 light chain, can
bind to a CD40
antigen. The anti-CD40 light chain can also be expressed with its
corresponding anti-CD40
heavy chain or fragment thereof to form an anti-CD40 antibody or fragment
thereof. The anti-
CD40 antibody or fragment thereof can be purified, and can be combined with a
pharmaceutically acceptable carrier.
[0228] An anti-DEC-205 light chain can be expressed with its corresponding
anti-DEC-205
heavy chain or fragment thereof. The corresponding anti-DEC-205 heavy chain or
fragment
thereof can be a heavy chain or fragment that when paired with the anti-DEC-
205 light chain, can
bind to a DEC-205 antigen. The anti-DEC-205 light chain can also be expressed
with its
corresponding anti-DEC-205 heavy chain or fragment thereof to form an anti-DEC-
205 antibody
or fragment thereof. The anti-DEC-205 antibody or fragment thereof can be
purified, and can be
combined with a pharmaceutically acceptable carrier.
[0229] An anti-tumor antigen light chain can be expressed with an anti-tumor
antigen heavy
chain or fragment thereof. The anti-tumor antigen light chain can also
expressed with an anti-
tumor antigen heavy chain or fragment thereof to form an anti-tumor antigen
antibody or
88
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
fragment thereof. The anti-tumor antibody or fragment thereof can be purified,
and can be
combined with a pharmaceutically acceptable carrier.
[0230] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40
antibody can
be dacetuzumab.
[0231] A conjugate or antibody construct can comprise an antibody light chain.
A light chain can
be a light chain of an anti-CD40 antibody which can bind to a CD40 antigen. A
light chain of an
anti-CD40 antibody can be dacetuzumab.
[0232] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be an IgG4 isotype. A heavy chain of an anti-CD40
antibody can
be bleselumab.
[0233] A conjugate or antibody construct can comprise an antibody light chain.
A light chain can
be a light chain of an anti-CD40 antibody which can bind to a CD40 antigen. A
light chain of an
anti-CD40 antibody can be bleselumab.
[0234] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40
antibody can
be lucatumumab.
[0235] A conjugate or antibody construct can comprise an antibody light chain.
A light chain can
be a light chain of an anti-CD40 antibody which can bind to a CD40 antigen. A
light chain of an
anti-CD40 antibody can be lucatumumab.
[0236] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40
antibody can
be ADC-1013.
[0237] A conjugate or antibody construct can comprise an antibody light chain.
A light chain can
be a light chain of an anti-CD40 antibody which can bind to a CD40 antigen. A
light chain of an
anti-CD40 antibody can be ADC-1013.
[0238] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be the humanized rabbit antibody APX005.
89
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0239] A conjugate or antibody construct can comprise an antibody light chain.
A light chain can
be a light chain of an anti-CD40 antibody which can bind to a CD40 antigen. A
light chain of an
anti-CD40 antibody can be the humanized rabbit antibody APX005.
[0240] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be Chi Lob 7/4.
[0241] A conjugate or antibody construct can comprise an antibody light chain.
A light chain can
be a light chain of an anti-CD40 antibody which can bind to a CD40 antigen. A
light chain of an
anti-CD40 antibody can be Chi Lob 7/4.
[0242] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40
antibody can
be SBT-040-G1WT.
[0243] A conjugate or antibody construct can comprise an antibody heavy chain.
A heavy chain
can be a heavy chain of an anti-CD40 antibody which can bind to a CD40
antigen. A heavy chain
of an anti-CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40
antibody can
be SBT-040 VH-hIgG1 wt.
[0244] A heavy chain of an anti-CD40 antibody can be an IgG2 isotype. A heavy
chain of an
anti-CD40 antibody can be SBT-040-G2.
[0245] A conjugate or antibody construct can comprise an antibody with
modifications occurring
at least at one amino acid residue. Modifications can be substitutions,
additions, mutations,
deletions, or the like. An antibody modification can be an insertion of an
unnatural amino acid.
[0246] A conjugate or antibody construct can comprise a light chain of an
amino acid sequence
having at least one, two, three, four, five, six, seven, eight, nine or ten
modifications but not more
than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence
relative to the natural
or original amino acid sequence. A conjugate or antibody construct can
comprise a heavy chain
of an amino acid sequence having at least one, two, three, four, five, six,
seven, eight, nine or ten
modifications but not more than 40, 35, 30, 25, 20, 15 or 10 modifications of
the amino acid
sequence relative to the natural or original amino acid sequence. A heavy
chain can be the heavy
chain of an anti-CD40 antibody which can bind to the CD40 antigen.
[0247] A conjugate or antibody construct can comprise an Fc domain of an IgG1
isotype. A
conjugate or antibody construct can comprise an Fc domain of an IgG2 isotype.
A conjugate or
antibody construct can comprise an Fc domain of an IgG3 isotype. A conjugate
can comprise an
Fc domain of an IgG4 isotype. A conjugate or antibody construct can have a
hybrid isotype
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprising constant regions from two or more isotypes. A conjugate or antibody
construct can be
an anti-CD40 antibody, in which the anti-CD40 antibody can be a monoclonal
human antibody
comprising a wild-type sequence of an IgG1 isoform, in particular, at an Fc
region of the
antibody.
[0248] Conjugates and antibody constructs disclosed herein can be non-natural,
designed, and/or
engineered. Conjugates and antibody constructs disclosed herein can be non-
natural, designed,
and/or engineered scaffolds comprising an antigen binding domain. Conjugates
and antibody
constructs disclosed herein can be non-natural, designed, and/or engineered
antibodies.
Conjugates and antibody constructs can include monoclonal antibodies.
Conjugates and antibody
constructs can comprise human antibodies. Conjugates and antibody constructs
can comprise
humanized antibodies. Conjugates and antibody constructs can comprise
monoclonal humanized
antibodies. Conjugates and antibody constructs can comprise recombinant
antibodies.
[0249] The Kd for binding of the Fc domain to an Fc receptor of a conjugate or
antibody
construct as described herein can increase when the tumor antigen binding
domain is bound to its
tumor antigen as compared to the Kd for binding of the Fc domain to an Fc
receptor of a
conjugate or antibody construct as described herein when the tumor antigen
binding domain is
not bound to its tumor antigen. For example, a conjugate or antibody construct
as described
herein can have a Kd for binding of the Fc domain to an Fc receptor in the
presence of the
binding domain that binds to a molecule on an immune cell, such as an antigen
presenting cell,
and the tumor targeting binding domain when the tumor targeting binding domain
is bound to its
tumor antigen that can be greater than or greater than about 100 nM. The Kd
for binding of the
Fc domain to an Fc receptor in the presence of the binding domain that binds
to a molecule on an
immune cell, such as an antigen presenting cell, and the tumor targeting
binding domain when
the tumor targeting binding domain is bound to its tumor antigen can be or can
be about 100 nM,
200 nM, 300 nM, 400 nM, 500 nM, or 1000 nM. The Kd for binding of the Fc
domain to an Fc
receptor in the presence of the binding domain that binds to a molecule on an
immune cell, such
as an antigen presenting cell, and the tumor targeting binding domain when the
tumor targeting
binding domain is bound to its tumor antigen can be from 100 nM to 200 nM, 100
nM to 300 nM,
100 nM to 400 nM, 100 nM to 500 nM, or 100 nM to 1000 nM. Additionally, the
conjugate or
antibody construct as described herein can have a Kd for binding of the Fc
domain to an Fc
receptor in the presence of the binding domain that binds to a molecule on an
immune cell, such
as an antigen presenting cell, and a tumor antigen binding domain when the
tumor antigen
binding domain is not bound to the tumor antigen is no greater than about
100nM and is no
greater than about 100 times a Kd for binding of the Fc domain to the Fc
receptor in an absence
91
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
of the binding domain that binds to a molecule on an immune cell, such as an
antigen presenting
cell, and a tumor antigen binding domain.
[0250] The Kd for binding of the binding domain that binds to a molecule on an
immune cell,
such as an antigen presenting cell, of a conjugate or antibody construct as
described herein can
increase when the tumor antigen binding domain is bound to its tumor antigen
as compared to the
Kd for binding of the binding domain that binds to a molecule on an immune
cell, such as an
antigen presenting cell, of a conjugate or antibody construct as described
herein when the tumor
antigen binding domain is not bound to its tumor antigen. For example, a
conjugate or antibody
construct as described herein can comprise a Kd for binding of the binding
domain that binds to a
molecule on an immune cell, such as an antigen presenting cell, when the tumor
antigen binding
domain is bound to its tumor antigen can be greater than or greater than about
100nM. The Kd
for binding of the binding domain that binds to a molecule on an immune cell,
such as an antigen
presenting cell, when the tumor antigen binding domain is bound to its tumor
antigen can be or
can be about 100nM, 200 nM, 300 nM, 400 nM, 500 nM, or 1000 nM. Kd for binding
of the
binding domain that binds to a molecule on an immune cell, such as an antigen
presenting cell,
when the tumor antigen binding domain is bound to its tumor antigen can be
from 100nM to 200
nM, 100 nM to 300 nM, 100 nM to 400 nM, 100 nM to 500 nM, or 100 nM to 1000
nM.
[0251] The effect of the tumor antigen binding domain and the binding domain
that binds to a
molecule on the immune cell, such as an antigen presenting cell, together can
be to cluster the
conjugates or antibody constructs on cells expressing tumor antigen, and thus
clustering immune
cells such as anantigen presenting cells around cancerous cells and at tumor
sites resulting in
activation of the immune cell effector functions or antigen presenting cell
effector functions. This
can include the activation of the molecule on the immune cell, such as an
antigen presenting cell,
when a bispecific tumor targeting antibody construct or conjugate is bound to
its tumor antigen,
such as activation of CD40, DEC-205, CD36 mannose scavenger receptor 1, DC-
SIGN,
CLEC9A, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin
2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B, or CD47. This can
include the activation of the molecule on the immune cell, such as an antigen
presenting cell,
when a bispecific tumor targeting antibody construct or conjugate is bound to
its tumor antigen,
such as activation of CD40, DEC-205, CD36 mannose scavenger receptor 1, DC-
SIGN,
CLEC9A, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin
2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B, CD47, TNFR2, or
TREM2. In some embodiments, this activation of the molecule on the immune
cell, such as an
antigen presenting cell, only occurs when the bispecific tumor targeting
antibody construct or
92
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
conjugate is bound to its tumor antigen. An immune cell effector function or
antigen presenting
cell effector function can include antibody dependent cellular cytotoxicity
(ADCC) of the tumor
antigen expressing cell, which can occur when the bispecific tumor targeting
conjugate is bound
to its tumor antigen. In some embodiments, ADCC of the tumor antigen
expressing cell only
occurs with the bispecific tumor targeting antibody construct or conjugate is
bound to its tumor
antigen. An immune cell effector function or antigen presenting cell effector
function can include
antibody dependent cellular phagocytosis (ADCP) of the tumor antigen
expressing cell, which
can occur when the bispecific tumor targeting conjugate is bound to its tumor
antigen. In some
embodiments, ADCP of the tumor antigen expressing cell only occurs with the
bispecific tumor
targeting antibody construct or conjugate is bound to its tumor antigen. In
certain embodiments, a
bispecific tumor targeting antibody construct or conjugate density of greater
than 1000, 2000,
3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more per cell, resulting
from the bispecific
tumor targeting antibody construct or conjugate binding to the tumor antigen,
induces signaling
in the immune cell such as an antigen presenting cell. Signaling can suitably
be measured in vitro
using a cell line expressing the tumor antigen bound by the target antigen
binding domain, and
primary antigen presenting cells or other immune cells isolated from a human
subject. Signaling
can be assessed as cytokine release, chemokine release, or increased
expression of cell surface
markers. Cytokine release can be measured by a cytokine release assay.
Chemokine release can
be measured by an ELISA immunoassay. Expression of cell surface markers can be
measured by
Fluorescent-Activated Cell Sorting (FACS). In certain embodiments, a
bispecific tumor targeting
conjugate density of greater than 1000, 2000, 3000, 4000, 5000, 6000, 7000,
8000, 9000, 10,000
or more per cell, resulting from the bispecific tumor targeting antibody
construct or conjugate
binding to the tumor antigen, induces ADCC of the cells expressing tumor
antigen. ADCC can
suitably be measured in vitro using a cell line expressing the tumor antigen
bound by the target
antigen binding domain, and cells such as NK cells and/or macrophages isolated
from a human
subject. ADCC can be determined by the frequency of remaining tumor antigen
expressing cells
in the co-culture. ADCP can be measured by an ADCP assay, which can be
determined by the
frequency of remaining tumor antigen expressing cells in the co-culture.
[0252] In some embodiments, the bispecific tumor targeting antibody constructs
or conjugates as
described herein can specifically bind to a tumor antigen in a cluster of
bispecific tumor targeting
antibody constructs or conjugates, and this clustering can induce a signal in
an immune cell such
as anantigen presenting cell. The bispecific tumor targeting antibody
constructs or conjugates as
described herein can specifically bind to a tumor antigen in a cluster of
bispecific tumor targeting
antibody constructs or conjugates, and this clustering can induce antibody
dependent cellular
93
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cytotoxicity. The bispecific tumor targeting antibody constructs or conjugates
as described herein
can specifically bind to a tumor antigen in a cluster of bispecific tumor
targeting antibody
constructs or conjugates and this clustering can result in an increased
avidity for a molecule on an
immune cell such as an antigen presenting cell. The bispecific tumor targeting
antibody
constructs or conjugates as described herein can specifically bind to a tumor
antigen in a cluster
of bispecific tumor targeting antibody constructs or conjugates and this
clustering can result in an
increased avidity of the Fc domain for an Fc receptor.
[0253] Sequences that can be used to produce antibodies for the antibody
constructs and
conjugates can comprise leader sequences. Leader sequences can be signal
sequences. Leader
sequences useful with the antibody constructs and conjugates and methods
described herein can
include, but are not limited to, an amino acid sequence comprising SEQ ID NO:
847, SEQ ID
NO: 848, and SEQ ID NO: 849.
[0254] A binding domain of a antibody construct or conjugate can be selected
in order to
recognize an antigen or molecule. For example, an antigen can be a cell
surface marker on target
cells associated with a disease or condition. An antigen can be expressed on
an immune cell. An
antigen can be a peptide or fragment thereof. An antigen can be expressed on
an antigen
presenting cell. An antigen can be expressed on a T cell, NK cell, NKT cell,
dendritic cell, a
macrophage, or a B cell. An antigen on an immune cell, such as an antigen
presenting cell, can be
a cell lineage marker or a cell surface protein expressed preferentially on
immune cells such as an
antigen presenting cells or a subset of immune or antigen presenting cells. An
antigen can be a
peptide presented in a major histocompatibility complex by cell. As another
example, a cell
surface marker recognized by the antigen binding domain can include
macromolecules associated
with viral and bacterial diseases or infections, autoimmune diseases and
cancerous diseases. An
antigen can be CD40 and an antigen binding domain can recognize a CD40
antigen.
[0255] An antigen can be a tumor antigen or fragment thereof. A tumor antigen
can be GD2,
GD3, GM2, Ley, polysialic acid, fucosyl GM1, GM3, Tn, STn, sLe(animal), or
GloboH. A tumor
antigen can be any antigen listed on tumor antigen databases, such as
TANTIGEN, or peptide
databases for T cell-defined tumor antigens, such as the Cancer Immunity
Peptide database. A
tumor antigen can also be any antigen listed in the review by Chen (Chen,
Cancer Immun 2004
[updated 2004 Mar 10; cited 2004 Apr 1]). Note that the 'antibody' can
recognize the 'tumor
antigen' or a peptide derived thereof, bound to an MHC molecule. An antigen
can be or can be at
least 80% homologous to an amino acid sequence of CD5, CD19, CD20, CD25, CD37,
CD30,
CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLA-DR,
carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, folate-binding protein,
A33, G250,
94
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
prostate-specific membrane antigen (PSMA), ferritin, CA-125, CA19-9, epidermal
growth factor,
p185HER2, IL-2 receptor, EGFRvIII (de2-7), EGFR, fibroblast activation
protein, tenascin, a
metalloproteinase, endosialin, vascular endothelial growth factor, avf33, WT1,
LMP2, HPV E6,
HPV E7, Her-2/neu, p53 nonmutant, NY-ESO-1, MelanA/MART1, Ras mutant, gp100,
p53
mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a Sarcoma
translocation breakpoint
fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen
receptor,
cyclin Bl, MYCN, RhoC, TRP-2, mesothelin, PSCA, MAGE Al, MAGE A3, CYP1B1,
PLAV1,
BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, Carbonic anhydrase IX, PAX5, 0Y-TES1,
Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3,
PAGE4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4,
MAGE C2, GAGE, EGFR, CMET, HER3, MUC1, CA6, NAPI2B, TROP2, CLDN6, CLDN16,
CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, UPK1B, VTCN1 (B7-H4), STRA6, TMPRSS3,
TMRRSS4, TMEM238, Clorf186, LIV1, ROR1, Fos-related antigen 1, VEGFR1,
endoglin,
VISTA, LLRC15, or a fragment thereof An antigen binding domain can be capable
of
recognizing a single antigen. An antigen binding domain can be capable of
recognizing two or
more different antigens.
[0256] An antibody construct or conjugate can comprise an Fc region with an Fc
domain. An Fc
domain is a structure that can bind to Fc receptors. An antibody construct or
conjugate can
comprise an Fc domain. Fc domains can be bound by Fc receptors (FcRs). Fc
domains can be
from antibodies. An Fc domain can be at least 80% homologous to an Fc domain
from an
antibody. An Fc region can be in a scaffold. An Fc region with an Fc domain
can be in an
antibody scaffold. An Fc region with an Fc domain can be in a non-antibody
scaffold. An
antibody construct or conjugate can comprise an Fc region with an Fc domain in
an antibody
scaffold. An antibody construct or conjugate can comprise an Fc region with an
Fc domain in a
non-antibody scaffold. An Fc domain can be in a scaffold. An Fc domain can be
in an antibody
scaffold. An Fc domain can be in a non-antibody scaffold. An antibody
construct or conjugate
can comprise an Fc domain in an antibody scaffold. An antibody construct or
conjugate can
comprise an Fc domain in a non-antibody scaffold. Fc domains of antibodies,
including those of
the present disclosure, can be bound by Fc receptors (FcRs). Fc domains can be
a portion of the
Fc region of an antibody. FcRs can bind to an Fc domain of an antibody. FcRs
can bind to an Fc
domain of an antibody bound to an antigen. FcRs can be organized into classes
(e.g., gamma (y),
alpha (a) and epsilon (6)) based on the class of antibody that the FcR
recognizes. The FcaR class
can bind to IgA and includes several isoforms, FcaRI (CD89) and Fcai.t.R. The
FcyR class can
bind to IgG and includes several isoforms, FcyRI (CD64), FcyRIIA (CD32a),
FcyRIIB (CD32b),
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
FcyRIIIA (CD16a), and FcyRIIIB (CD16b). An FcyRIIIA (CD16a) can be an FcyRIIIA
(CD16a)
F158 variant. An FcyRIIIA (CD16a) can be an FcyRIIIA (CD16a) V158 variant.
Each FcyR
isoform can differ in affinity to the Fc region of the IgG antibody. For
example, FcyRI can bind
to IgG with greater affinity than FcyRII or FcyRIII. The affinity of a
particular FcyR isoform to
IgG can be controlled, in part, by a glycan (e.g., oligosacccharaide) at
position CH2 84.4 of the
IgG antibody. For example, fucose containing CH2 84.4 glycans can reduce IgG
affinity for
FcyRIIIA. In addition, GO glucans can have increased affinity for FcyRIIIA due
to the lack of
galactose and terminal GlcNAc moiety.
[0257] Binding of an Fc domain to an FcR can enhance an immune response. FcR-
mediated
signaling that can result from an Fc region binding to an FcR can lead to the
maturation of
immune cells. FcR-mediated signaling that can result from an Fc domain binding
to an FcR can
lead to the maturation of dendritic cells. FcR-mediated signaling that can
result from an Fc
domain binding to an FcR can lead to antibody dependent cellular cytotoxicity.
FcR-mediated
signaling that can result from an Fc domain binding to an FcR can lead to more
efficient immune
cell antigen uptake and processing. FcR-mediated signaling that can result
from an Fc region
binding to an FcR can lead to more efficient dendritic cell antigen uptake and
processing. FcR-
mediated signaling that can result from an Fc region binding to an FcR can
increase antigen
presentation. FcR-mediated signaling that can result from an Fc region binding
to an FcR can
increase antigen presentation by immune cells. FcR-mediated signaling that can
result from an Fc
region binding to an FcR can increase antigen presentation by antigen
presenting cells. FcR-
mediated signaling that can result from an Fc domain binding to an FcR can
increase antigen
presentation by dendritic cells. FcR-mediated signaling that can result from
an Fc domain
binding to an FcR can promote the expansion and activation of T cells. FcR-
mediated signaling
that can result from an Fc domain binding to an FcR can promote the expansion
and activation of
CD8+ T cells. FcR-mediated signaling that can result from an Fc domain binding
to an FcR can
influence immune cell regulation of T cell responses. FcR-mediated signaling
that can result
from an Fc domain binding to an FcR can influence immune cell regulation of T
cell responses.
FcR-mediated signaling that can result from an Fc domain binding to an FcR can
influence
dendritic cell regulation of T cell responses. FcR-mediated signaling that can
result from an Fc
domain binding to an FcR can influence functional polarization of T cells
(e.g., polarization can
be toward a TH1 cell response).
[0258] The profile of FcRs on a DC can impact the ability of the DC to respond
upon stimulation.
For example, most DC can express both CD32A and CD32B, which can have opposing
effects
on IgG-mediated maturation and function of DCs: binding of IgG to CD32A can
mature and
96
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
activate DCs in contrast with CD32B, which can mediate inhibition due to
phosphorylation of
immunoreceptor tyrosine-based inhibition motif (ITIM), after CD32B binding of
IgG. Therefore,
the activity of these two receptors can establish a threshold of DC
activation. Furthermore,
difference in functional avidity of these receptors for IgG can shift their
functional balance.
Hence, altering the Fc domain binding to FcRs can also shift their functional
balance, allowing
for manipulation (either enhanced activity or enhanced inhibition) of the DC
immune response.
[0259] A modification in the amino acid sequence Fc domain can alter the
recognition of an FcR
for the Fc domain. However, such modifications can still allow for FcR-
mediated signaling. A
modification can be a substitution of an amino acid at a residue (e.g.,
wildtype) for a different
amino acid at that residue. A modification can permit binding of an FcR to a
site on the Fc region
that the FcR may not otherwise bind to. A modification can increase binding
affinity of an FcR to
the Fc domain that the FcR may have reduced binding affinity for. A
modification can decrease
binding affinity of an FcR to a site on the Fc domain that the FcR may have
increased binding
affinity for. A modification can increase the subsequent FcR-mediated
signaling after Fc binding
to an FcR.
[0260] An antibody construct or conjugate can comprise an Fc region with at
least one amino
acid change as compared to the sequence of the wild-type Fc region. An
antibody construct or
conjugate can comprise an Fc domain with at least one amino acid change as
compared to the
sequence of the wild-type Fc domain. An amino acid change in an Fc region can
allow the
antibody construct or conjugate to bind to at least one Fc receptor with
greater affinity compared
to a wild-type Fc region. An amino acid change in an Fc domain can allow the
antibody to bind
to at least one Fc receptor with greater affinity compared to a wild-type Fc
domain. An Fc region
can comprise an amino acid sequence having at least one, two, three, four,
five, six, seven, eight,
nine or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10
modifications of the
amino acid sequence relative to the natural or original amino acid sequence.
An Fc domain can
comprise an amino acid sequence having at least one, two, three, four, five,
six, seven, eight, nine
or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10
modifications of the amino
acid sequence relative to the natural or original amino acid sequence. An Fc
region can be an Fc
region of an anti-CD40 antibody. An Fc domain can be an Fc domain of an anti-
CD40 antibody.
An Fc region can contain an Fc domain. An Fc region can be an Fc domain.
[0261] An antibody construct or conjugate can comprise an antibody comprising
a sequence of
the IgG1 isoform that has been modified from the wildtype IgG1 sequence. A
modification can
comprise a substitution at one or more one amino acid residues of an Fc domain
such as at 5
different amino acid residues including L235V/F243L/R292P/Y300L/P396L
(IgG1VLPLL). The
97
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
numbering of amino acids residues described herein can be according to the EU
index. This
modification can be located in a portion of an antibody sequence which can
encode an Fc region
of the antibody and in particular, can be located in portions of the Fc region
that can bind to Fc
receptors (i.e., the Fc domain). A modification can comprise a substitution at
one or more amino
acid residues such as at 2 different amino acid residues of an Fc domain
including S239D/I332E
(IgG1DE). This modification can be located in a portion of an antibody
sequence which encodes
an Fc region of the antibody and in particular, are located in portions of the
Fc region that can
bind to Fc receptors (i.e., the Fc domain). A modification can comprise a
substitution at one or
more amino acid residues such as at 3 different amino acid residues of an Fc
domain including
S298A/E333A/K334A (IgGlAAA). The modification can be located in a portion of
an antibody
sequence which can encode an Fc region of the antibody and in particular, can
be located in
portions of the Fc region that can bind Fc receptors (i.e., the Fc domain).
[0262] An antibody construct or conjugate can comprise a monoclonal anti-CD40
human
antibody comprising a sequence of the IgG1 isoform that has been modified from
the wildtype
IgG1 sequence. A modification can comprise a substitution at one or more one
amino acid
residues such as at 5 different amino acid residues of an Fc domain including
L235V/F243L/R292P/Y300L/P396L (SBT-040-G1VLPLL). The numbering of amino acids
residues described herein can be according to the EU index. This modification
can be located in a
portion of an antibody sequence which can encode an Fc region of the antibody
and in particular,
can be located in portions of the Fc region that can bind to Fc receptors
(i.e., the Fc domain). A
modification can comprise a substitution at one or more amino acid residues
such as at 2 different
amino acid residues of an Fc domain including S239D/I332E (SBT-040-G1DE). This
modification can be located in a portion of an antibody sequence which encodes
an Fc region of
the antibody and in particular, are located in portions of the Fc region that
can bind to Fc
receptors (i.e., the Fc domain). A modification can comprise a substitution at
one or more one
amino acid residues such as at 3 different amino acid residues of an Fc domain
including
S298A/E333A/K334A (SBT-040-G1AAA). This modification can be located in a
portion of an
antibody sequence which can encode an Fc region of the antibody and in
particular, can be
located in portions of the Fc region that can bind Fc receptors (i.e., the Fc
domain).
[0263] Binding of Fc receptors to an Fc region can be affected by amino acid
substitutions. For
example, SBT-040-VLPLL is an antibody with an amino acid sequence of a heavy
chain of
human anti-CD40 monoclonal antibody with modifications to a wild-type IgG1 Fc
domain
(L235V/F243L/R292P/Y300L/P396L). Binding of some Fc receptors to the Fc region
of SBT-
040-VLPLL can be enhanced compared to wild-type by as result of the
98
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
L235V/F243L/R292P/Y300L/P396L amino acid modifications. However, binding of
other Fc
receptors to the Fc region of SBT-040-VLPLL can be reduced compared to wild-
type by the
L235V/F243L/R292P/Y300L/P396L amino acid modifications. For example, the
binding
affinities of SBT-040-VLPLL to FcyRIIIA and to FcyRIIA can be enhanced
compared to wild-
type whereas the binding affinity of SBT-040-VLPLL to FcyRIIB can be reduced
compared to
wild-type. SBT-040-DE antibody is an antibody with an amino acid sequence of a
heavy chain of
human anti-CD40 monoclonal antibody with modifications to a wild-type IgG1 Fc
domain
(S239D/I332E). Binding of Fc receptors to the Fc region of SBT-040-DE can be
enhanced
compared to wild-type as a result of the S239D/I332E amino acid modification.
However,
binding of some Fc receptors to the Fc region of SBT-040-G1DE can be reduced
compared to
wild-type by S239D/I332E amino acid modification. For example, the binding
affinities of SBT-
040-DE to FcyRIIIA and to FcyRIIB can be enhanced compared to wild-type.
Binding of Fc
receptors to an Fc region of are affected by amino acid substitutions. SBT-040-
G1AAA antibody
is an antibody with an amino acid sequence of a heavy chain of a human anti-
CD40 monoclonal
antibody with modifications to a wild-type IgG1 Fc domain (S298A/E333A/K334A).
Binding of
Fc receptors to an Fc region of SBT-040-G1AAA can be enhanced compared to wild-
type as a
result of the S298A/E333A/K334A amino acid modification. However, binding of
some Fc
receptors to the Fc region of SBT-040-G1AAA can be reduced compared to wild-
type by
S298A/E333A/K334A amino acid modification. Binding affinities of SBT-040-G1AAA
to
FcyRIIIA can be enhanced compared to wild-type whereas the binding affinity of
SBT-040-
G1AAA to FcyRIIB can be reduced compared to wildtype.
[0264] In some embodiments, the heavy chain of a human IgG2 antibody can be
mutated at
cysteines as positions 127, 232, or 233. In some embodiments, the light chain
of a human IgG2
antibody can be mutated at a cysteine at position 214. The mutations in the
heavy and light
chains of the human IgG2 antibody can be from a cysteine residue to a serine
residue.
[0265] While an antibody construct or conjugate of the present disclosure can
comprise a first
binding domain and a second binding domain (or, in some cases, a third binding
domain) with
wild-type or modified amino acid sequences encoding the Fc region or Fc
domain, the
modifications of the Fc region or the Fc domain from the wild-type sequence
may not
significantly alter binding and/or affinity of the binding domains. For
example, binding and/or
affinity of an antibody construct or conjugate comprising a first binding
domain and a second
binding domain (or, in some cases, a third binding domain) and having the Fc
domain
modifications of SBT-040-G1WT, SBT-040-G1VLPLL, SBT-040-G1DE, or SBT-040-G1AAA
may not be significantly altered by modification of an Fc region or Fc domain
amino acid
99
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
sequence compared to a wild-type sequence. Modifications of an Fc region or Fc
domain from a
wild-type sequence may not alter binding and/or affinity of a first binding
domain that binds, for
example, to CD40 or DEC-205. Additionally, the binding and/or affinity of the
binding domains
described herein, for example a first binding domain, a second binding domain
(or, in some cases,
a third binding domain), and an Fc domain modification selected from SBT-040-
G1WT, SBT-
040-G1VLPLL, SBT-040-G1DE, and SBT-040-G1AAA, may be comparable to the binding
and/or affinity of wild-type antibodies.
In some embodiments, a Kd for binding of the first binding domain to the tumor
antigen in the
presence of the immune-stimulatory compound is no greater than about two
times, five times, ten
times, or fifty times a Kd for binding of the first binding domain to the
tumor antigen in an
absence of the immune-stimulatory compound. In some embodiments, a Kd for
binding of the
second binding domain to the antigen on the antigen presenting cell in the
presence of the
immune-stimulatory compound is no greater than about two times, five times,
ten times, or fifty
times a Kd for binding of the second binding domain to the antigen on the
antigen presenting cell
in an absence of the immune-stimulatory compound. In some embodiments, a Kd
for binding of
the first binding domain to the tumor antigen is no greater than about 100 nM.
In some
embodiments, a Kd for binding of the second binding domain to the antigen on
an antigen
presenting cell is no greater than about 100 nM.
[0266] In some embodiments, a Kd for binding of the Fc domain to the Fc
receptor in the
presence of the immune-stimulatory compound is no greater than about two
times, five times, ten
times, or fifty times a Kd for binding of the Fc domain to the Fc receptor in
an absence of the
immune-stimulatory compound.
[0267] In some embodiments, the Fc domain is an Fc domain variant comprising
at least one
amino acid residue change as compared to a wild type sequence of the Fc
domain.
[0268] In some embodiments, the Fc domain variant binds to an Fc receptor with
altered affinity
as compared to the wild type Fc domain.
[0269] In some embodiments, the at least one amino acid residue change is
selected from a group
consisting of: a) F243L, R292P, Y300L, L235V, and P396L, wherein numbering of
amino acid
residues in the Fc domain is according to the EU index; b) S239D and 1332E,
wherein numbering
of amino acid residues in the Fc domain is according to the EU index; and c)
S298A, E333A, and
K334A, wherein numbering of amino acid residues in the Fc domain is according
to the EU
index.
[0270] In some embodiments, the antibody construct or conjugate induces
secretion of cytokines
by an immune cell as measured by a cytokine release assay. In some
embodiments, the cytokine
100
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
is IFN-y, IL-8, IL-12, IL-2, or a combination thereof. In some embodiments,
the antibody
construct or conjugate induces antigen presentation on a dendritic cell, B
cell, macrophage, or a
combination thereof.
TABLE 3. Exemplary Tumor Antibody CDRs
Antibody Region SEQ ID NO: Antibody Region SEQ
ID NO:
Nimotuzumab HCDR1 52 HCDR3 158
HCDR2 53 LCDR1 161
HCDR3 54 LCDR2 162
LCDR1 57 LCDR3 163
LCDR2 58 huDS6 HCDR1 169
LCDR3 59 HCDR2 170
Zalutumumab HCDR1 65 HCDR3 171
HCDR2 66 LCDR1 174
HCDR3 67 LCDR2 175
LCDR1 70 LCDR3 176
LCDR2 71 Lifastuzumab HCDR1 182
LCDR3 72 HCDR2 183
Onartuzumab HCDR1 78 HCDR3 184
HCDR2 79 LCDR1 187
HCDR3 80 LCDR2 188
LCDR1 83 LCDR3 189
LCDR2 84 Sacituzumab HCDR1 195
LCDR3 85 HCDR2 196
Patritumab HCDR1 91 HCDR3 197
HCDR2 92 LCDR1 200
HCDR3 93 LCDR2 201
LCDR1 96 LCDR3 202
LCDR2 97 PR1A3 HCDR1 208
LCDR3 98 HCDR2 209
Clivatuzumab HCDR1 104 HCDR3 210
HCDR2 105 LCDR1 213
HCDR3 106 LCDR2 214
LCDR1 109 LCDR3 215
LCDR2 110 Humanized PR1A3 HCDR1 805
LCDR3 111 HCDR2 806
Sofituzumab HCDR1 117 HCDR3 807
HCDR2 118 LCDR1 808
HCDR3 119 LCDR2 809
LCDR1 122 LCDR3 810
LCDR2 123 Humanized Ab2-3 HCDR1 823
LCDR3 124 HCDR2 824
Edrecolomab HCDR1 130 HCDR3 825
HCDR2 131 LCDR1 826
HCDR3 132 LCDR2 827
LCDR1 135 LCDR3 828
LCDR2 136 IMAB362, HCDR1 221
LCDR3 137 Claudiximab HCDR2 222
Adecatumumab HCDR1 143 HCDR3 223
HCDR2 144 LCDR1 226
HCDR3 145 LCDR2 227
LCDR1 148 LCDR3 228
LCDR2 149 AMG595 HCDR1 260
LCDR3 150 HCDR2 261
Anetumab HCDR1 156 HCDR3 262
HCDR2 157 LCDR1 265
101
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Antibody Region SEQ ID NO: Antibody Region SEQ
ID NO:
LCDR2 266 HCDR2 391
LCDR3 267 HCDR3 392
ABT806 HCDR1 273 LCDR1 395
HCDR2 274 LCDR2 396
HCDR3 275 LCDR3 397
LCDR1 278 J591 variant 2 HCDR1 403
LCDR2 279 HCDR2 404
LCDR3 280 HCDR3 405
Sibrotuzumab HCDR1 286 LCDR1 408
HCDR2 287 LCDR2 409
HCDR3 288 LCDR3 410
LCDR1 291 Rovalpituzumab HCDR1 416
LCDR2 292 HCDR2 417
LCDR3 293 HCDR3 418
DS-8895a variant 1 HCDR1 299 LCDR1 421
HCDR2 300 LCDR2 422
HCDR3 301 LCDR3 423
LCDR1 304 PF-06647020 HCDR1 429
LCDR2 305 HCDR2 430
LCDR3 306 HCDR3 431
DS-8895a variant 2 HCDR1 312 LCDR1 434
HCDR2 313 LCDR2 435
HCDR3 314 LCDR3 436
LCDR1 317 Anti-PTK7 HCDR1 442
LCDR2 318 HCDR2 443
LCDR3 319 HCDR3 444
Anti-EphA2 HCDR1 325 LCDR1 447
HCDR2 326 LCDR2 448
HCDR3 327 LCDR3 449
LCDR1 330 Ladiratuzumab HCDR1 455
LCDR2 331 HCDR2 456
LCDR3 332 HCDR3 457
Narnatumab HCDR1 338 LCDR1 460
HCDR2 339 LCDR2 461
HCDR3 340 LCDR3 462
LCDR1 343 Cirmtuzumab HCDR1 468
LCDR2 344 HCDR2 469
LCDR3 345 HCDR3 470
RG7841 HCDR1 351 LCDR1 473
HCDR2 352 LCDR2 474
HCDR3 353 LCDR3 475
LCDR1 356 anti-MAGE-A3 HCDR1 481
LCDR2 357 HCDR2 482
LCDR3 358 HCDR3 483
Farletuzumab HCDR1 364 LCDR1 486
HCDR2 365 LCDR2 487
HCDR3 366 LCDR3 488
LCDR1 369 Anti-NY-ESO-1 HCDR1 494
LCDR2 370 HCDR2 495
LCDR3 371 HCDR3 496
Mirvetuximab HCDR1 377 LCDR1 499
HCDR2 378 LCDR2 500
HCDR3 379 LCDR3 501
LCDR1 382
LCDR2 383
LCDR3 384
J591 variant 1 HCDR1 390
102
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
TABLE 4. Additional Exemplary Tumor Antibody CDRs
Antibody Region SEQ ID NO:
Pertuzumab HCDR1 13
HCDR2 14
HCDR3 15
LCDR1 18
LCDR2 19
LCDR3 20
Trastuzumab HCDR1 673
HCDR2 674
HCDR3 675
LCDR1 676
LCDR2 677
LCDR3 678
Cetuximab HCDR1 26
HCDR2 27
HCDR3 28
LCDR1 31
LCDR2 32
LCDR3 33
Panitumumab HCDR1 39
HCDR2 40
HCDR3 41
LCDR1 44
LCDR2 45
LCDR3 46
TABLE 5. Tumor Antibody VH sequences and VL sequences
Antibody Region SEQ ID NO:
Nimotuzumab VH 51 Humanized Ab2-3 VH 829
VL 56 VL 830
Zalutumumab VH 64 IMAB362, VH 220
VL 69 Claudiximab VL 225
Onartuzumab VH 77 AMG595 VH 259
VL 82 VL 264
Patritumab VH 90 ABT806 VH 272
VL 95 VL 277
Clivatuzumab VH 103 Sibrotuzumab VH 285
VL 108 VL 290
Sofituzumab VH 116 DS-8895a variant 1 VH 298
VL 121 VL 303
Edrecolomab VH 129 DS-8895a variant 2 VH 311
VL 134 VL 316
Adecatumumab VH 142 Anti-EphA2 VH 324
VL 147 VL 329
Anetumab VH 155 Narnatumab VH 337
VL 160 VL 342
huDS6 VH 168 RG7841 VH 350
VL 173 VL 355
Lifastuzumab VH 181 Farletuzumab VH 363
VL 186 VL 368
Sacituzumab VH 194 Mirvetuximab VH 376
VL 199 VL 381
PR1A3 VH 207 J591 variant 1 VH
389
VL 212 VL 394
Humanized PR1A3 VH 811 J591 variant 2 VH
402
VL 812 VL 407
103
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Rovalpituzumab VH 415 VL 459
VL 420 Cirmtuzumab VH 467
PF-06647020 VH 428 VL 472
VL 433 Anti-MAGE-A3 VH 480
Anti-PTK7 VH 441 VL 485
VL 446 Anti-NY-ESO-1 VH 493
Ladiratuzumab VH 454 VL 498
TABLE 6. Additional Exemplary Tumor Antibody VH sequences and VL sequences
Antibody Region SEQ ID NO:
Pertuzumab VH 12
VL 17
Cetuximab VH 25
VL 30
Panitumumab VH 38
VL 43
VL 498
Trastuzumab VH 679
VL 680
104
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
TABLE 7. Exemplary Tumor Antibody Heavy Chain (HC) and Light Chain (LC)
sequences
Antibody Region SEQ ID NO:
Nimotuzumab HC 50 LC 289
LC 55 DS-8895a variant 1 HC 297
Zalutumumab HC 63 LC 302
LC 68 DS-8895a variant 2 HC 310
Onartuzumab HC 76 LC 315
LC 81 Anti-EphA2 HC 323
Patritumab HC 89 LC 328
LC 94 Narnatumab HC 336
Clivatuzumab HC 102 LC 341
LC 107 RG7841 HC 349
Sofituzumab HC 115 LC 354
LC 120 Farletuzumab HC 362
Edrecolomab HC 128 LC 367
LC 133 Mirvetuximab HC 375
Adecatumumab HC 141 LC 380
LC 146 J591 variant 1 HC
388
Anetumab HC 154 LC 393
LC 159 J591 variant 2 HC 401
huDS6 HC 167 LC 406
LC 172 Rovalpituzumab HC 414
Lifastuzumab HC 180 LC 419
LC 185 PF-06647020 HC 427
Sacituzumab HC 193 LC 432
LC 198 Anti-PTK7 HC 440
PR1A3 HC 206 LC 445
LC 211 Ladiratuzumab HC 453
Humanized PR1A3 HC 813 LC 458
LC 814 Cirmtuzumab HC 466
Humanized Ab2-3 HC 831 LC 471
LC 832 Anti-MAGE-A3 HC 479
IMAB362, HC 219 LC 484
Claudiximab LC 224 Anti-NY-ESO-1 HC 492
AMG595 HC 258 LC 497
LC 263 LC 682
ABT806 HC 271
LC 276
Sibrotuzumab HC 284
TABLE 8. Additional Exemplary Tumor Antibody Heavy Chain (HC) and Light Chain
(LC)
sequences
Antibody Region SEQ ID NO:
Pertuzumab HC 11
LC 16
Cetuximab HC 24
LC 29
Panitumumab HC 37
LC 42
LC 263
Trastuzumab HC 681
LC 682
105
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
TABLE 9. Exemplary Fusion Sequences ¨ CD40 fusions via the heavy chain
Fusion SEQ ID NO:
HC CD40 mAb with Cetuximab ScFv 34
HC Cetuximab with CD40 mAb ScFv 35
HC CD40 mAb with Panitumumab ScFv 47
HC Panitumumab with CD40 mAb ScFv 48
HC CD40 mAb with Nimotuzumab ScFv 60
HC Nimotuzumab with CD40 mAb ScFv 61
HC CD40 mAb with Zalutumumab ScFv 73
HC Zalutumumab with CD40 mAb ScFv 74
HC CD40 mAb with Onartuzumab ScFv 86
HC Onartuzumab with CD40 mAb ScFv 87
HC CD40 mAb with Patritumab ScFv 99
HC Patritumab with CD40 mAb ScFv 100
HC CD40 mAb with Clivatuzumab ScFv 112
HC Clivatuzumab with CD40 mAb ScFv 113
HC CD40 mAb with Sofituzumab ScFv 125
HC Sofituzumab with CD40 mAb ScFv 126
HC CD40 mAb with Edrecolomab ScFv 138
HC Edrecolomab with CD40 mAb ScFv 139
HC CD40 mAb with Adecatumumab ScFv 151
HC Adecatumumab with CD40 mAb ScFv 152
HC CD40 mAb with Anetumab ScFv 164
HC Anetumab with CD40 mAb ScFv 165
HC CD40 mAb with huDS6 mAb ScFv 177
HC huDS6mAb with CD40 mAb ScFv 178
HC CD40 mAb with Lifastuzumab ScFv 190
HC Lifastuzumab with CD40 mAb ScFv 191
HC CD40 mAb with Sacituzumab ScFv 203
HC Sacituzumab with CD40 mAb ScFv 204
HC CD40 mAb with PR1A3 mAb ScFv 216
HC PR1A3mAb with CD40 mAb ScFv 217
HC CD40 mAb with Humanized PR1A3 mAb ScFv 815
HC Humanized PR1A3 mAb with CD40 mAb ScFv 816
HC Humanized PR1A3with CD40 mAb scFv (LH) 843
HC CD40 mAb with Humanized Ab2-3 mAb ScFv 833
HC Humanized Ab2-3 mAb with CD40 mAb ScFv 834
HC Humanized Ab2-3 mAb with CD40 mAb scFv (LH) 841
HC CD40 mAb with Claudiximab ScFv 229
HC Claudiximab with CD40 mAb ScFv 230
HC CD40 mAb with AMG595 mAb ScFv 268
HC AMG595 mAb with CD40 mAb ScFv 269
HC CD40 mAb with ABT806 mAb ScFv 281
HC ABT806 mAb with CD40 mAb ScFv 282
HC CD40 mAb with Sibrotuzumab mAb ScFv 294
HC Sibrotuzumab with CD40 mAb ScFv 295
HC CD40 mAb with DS-8895a variant 1 mAb ScFv 307
HC DS-8895a variant 1 mAb with CD40 mAb ScFv 308
HC CD40 mAb with DS-8895a variant 2 mAb ScFv 320
HC DS-8895a variant 2 mAb with CD40 mAb ScFv 321
HC CD40 mAb with Anti-EphA2 mAb ScFv 333
HC Anti-EphA2 mAb with CD40 mAb ScFv 334
HC CD40 mAb with Narnatumab mAb ScFv 346
106
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Fusion SEQ ID NO:
HC Narnatumab mAb with CD40 mAb ScFv 347
HC CD40 mAb with RG7841 mAb ScFv 359
HC RG7841 mAb with CD40 mAb ScFv 360
HC CD40 mAb with Farletuzumab mAb ScFv 372
HC Farletuzumab mAb with CD40 mAb ScFv 373
HC CD40 mAb with Mirvetuximab mAb ScFv 385
HC Mirvetuximab mAb with CD40 mAb ScFv 386
HC CD40 mAb with J591 variant 1 mAb ScFv 398
HC J591 variant 1 mAb with CD40 mAb ScFv 399
HC CD40 mAb with J591 variant 2 mAb ScFv 411
HC J591 variant 2 mAb with CD40 mAb ScFv 412
HC CD40 mAb with Rovalpituzumab mAb ScFv 424
HC Rovalpituzumab mAb with CD40 mAb ScFv 425
HC CD40 mAb with PF-06647020 mAb ScFv 437
HC PF-06647020 mAb with CD40 mAb ScFv 438
HC CD40 mAb with anti-PTK7 mAb ScFv 450
HC PTK7 mAb with CD40 mAb ScFv 451
HC CD40 mAb with mAb of Ladiratuzumab ScFv 463
HC Ladiratuzumab with CD40 mAb ScFv 464
HC CD40 mAb with Cirmtuzumab mAb ScFv 476
HC Cirmtuzumab mAb with CD40 mAb ScFv 477
HC CD40 mAb with anti-MAGE-A3 mAb ScFv 489
HC tumor mAb with CD40 mAb ScFv 490
HC CD40 mAb with anti-NY-ESO-1 mAb ScFv 502
HC anti-NY-ESO-1 mAb with CD40 mAb ScFv 503
TABLE 10. Additional Exemplary Fusion Sequences ¨ CD40 fusions via the heavy
chain
Fusion SEQ ID NO:
HC CD40 mAb with Pertuzumab ScFv 21
HC Pertuzumab with CD40 mAb ScFv 22
HC Pertuzumab with CD40 mAb scFv (LH) 845
HC CD40 mAb with Trastuzumab mAb ScFv 683
HC Trastuzumab mAb with CD40 mAb ScFv 684
HC CD40 mAb with Trastuzumab mAb ScFv 796
(LH,25mer)
TABLE 11. Exemplary APC Antibody CDRs
Antibody Region SEQ ID NO:
CP-870893 HCDR1 3
HCDR2 4
HCDR3 5
LCDR1 8
LCDR2 9
LCDR3 10
DEC-205 variant 1 HCDR1 234
HCDR2 235
HCDR3 236
LCDR1 239
LCDR2 240
LCDR3 241
DEC-205 variant 2 HCDR1 247
HCDR2 248
HCDR3 249
LCDR1 252
LCDR2 253
LCDR3 254
DC-SIGN variant HCDR1 640
1 HCDR2 641
107
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Antibody Region SEQ ID NO:
HCDR3 642
LCDR1 643
LCDR2 644
LCDR3 645
DC-SIGN variant HCDR1 646
2 HCDR2 647
HCDR3 648
LCDR1 649
LCDR2 650
LCDR3 651
DC-SIGN variant HCDR1 652
3 HCDR2 653
HCDR3 654
LCDR1 655
LCDR2 656
LCDR3 657
TABLE 12. Additional Exemplary APC Antibody CDRs
Antibody Region SEQ ID NO:
SBT-040 (G1/G2) HCDR1 3
HCDR2 4
HCDR3 5
LCDR1 8
LCDR2 9
LCDR3 10
Dacetuzumab HCDR1 582
HCDR2 583
HCDR3 584
LCDR1 587
LCDR2 588
LCDR3 589
Bleselumab HCDR1 592
HCDR2 593
HCDR3 594
LCDR1 597
LCDR2 598
LCDR3 599
lucatumumab HCDR1 602
HCDR2 603
HCDR3 604
LCDR1 607
LCDR2 608
LCDR3 609
ADC-1013 HCDR1 612
HCDR2 613
HCDR3 614
LCDR1 617
LCDR2 618
LCDR3 619
APX005 HCDR1 622
HCDR2 623
HCDR3 624
LCDR1 627
LCDR2 628
LCDR3 629
Chi Lob 7/4 HCDR1 632
HCDR2 633
108
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Antibody Region SEQ ID NO:
HCDR3 634
LCDR1 637
LCDR2 638
LCDR3 639
HCDR2 653
HCDR3 654
LCDR1 655
LCDR2 656
LCDR3 657
TABLE 13. Exemplary APC Antibody VH sequences and VL sequences
Antibody Region SEQ ID NO:
CP-870893 VH 2
VL 7
DEC-205 variant 1 VH 233
VL 238
DEC-205 variant 2 VH 246
VL 251
CD36 mannose VH 658
Scavenger VL 659
Receptor
CLEC9A VH 660
VL 661
TABLE 14. Additional Exemplary APC Antibody VH sequences and VL sequences
Antibody Region SEQ ID NO:
SBT-040 VH 2
VL 7
Dacetuzumab VH 581
VL 586
Bleselumab VH 591
VL 596
Lucatumumab VH 601
VL 606
ADC-1013 VH 611
VL 616
APX005 VH 621
VL 626
Chi Lob 7/4 VH 631
_ VL 636
TABLE 15. Exemplary APC Antibody Heavy Chain and Light Chain sequences
Antibody Region SEQ ID NO:
CP-870893 HC 1
LC 6
DEC-205 HC 232
(QVQLVQvariant LC 237
1)
DEC-205 HC 245
(variant 2) LC 250
CLEC12A HC 662
variant
1
HC 663
109
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Antibody Region SEQ ID NO:
variant
2
HC 664
variant
3
LC 665
BDCA-2 HC 666
Variant 1 LC 669
BDCA-2 HC 667
Variant 2 LC 670
BDCA-2 HC 668
Variant 3 LC 671
TABLE 16. Additional Exemplary APC Antibody Heavy Chain and Light Chain
sequences
Antibody Region SEQ ID NO:
Anti-CD40 HC 577
(IgG1)
HC 578
(IgG2)
LC 579
Dacetuzumab HC 580
LC 585
Bleselumab HC 590
LC 595
Lucatumumab HC 600
LC 605
ADC-1013 HC 610
LC 615
APX005 HC 620
LC 625
Chi Lob 7/4 HC 630
LC 635
TABLE 17. Exemplary Fusion Sequences ¨ DEC-205 fusions via the heavy chain
Fusion SEQ ID NO:
HC DEC-205 mAb with Pertuzumab mAb ScFv 505
HC Pertuzumab with DEC-205 mAb ScFv 506
LC Pertuzumab containing CD40 mab scFv (LH) 846
HC DEC-205 mAb with Cetuximab mAb ScFv 507
HC Cetuximab with DEC-205 mAb ScFv 508
HC DEC-205 mAb with Panitumumab mAb ScFv 509
HC Panitumumab with DEC-205 mAb ScFv 510
HC DEC-205 mAb with Nimotuzumab mAb ScFv 511
HC Nimotuzumab with DEC-205 mAb ScFv 512
HC DEC-205 mAb with Zalutumumab mAb ScFv 513
HC Zalutumumab with DEC-205 mAb ScFv 514
HC DEC-205 mAb with Onartuzumab mAb ScFv 515
HC Onartuzumab with DEC-205 mAb ScFv 516
HC DEC-205 mAb with Patritumab ScFv 517
HC Patritumab with DEC-205 mAb ScFv 518
HC DEC-205 mAb with Clivatuzumab ScFv 519
HC Clivatuzumab with DEC-205 mAb ScFv 520
HC DEC-205 mAb with Sofituzumab ScFv 521
HC Sofituzumab with DEC-205 mAb ScFv 522
HC DEC-205 mAb with Edrecolomab ScFv 523
HC Edrecolomab with DEC-205 mAb ScFv 524
110
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Fusion SEQ ID NO:
HC DEC-205 mAb with Adecatumumab ScFv 525
HC Adecatumumab with DEC-205 mAb ScFv 526
HC DEC-205 mAb with Anetumab ScFv 527
HC Anetumab with DEC-205 mAb ScFv 528
HC DEC-205 mAb with huDS6 mAb ScFv 529
HC huDS6 mAb with DEC-205 mAb ScFv 530
HC DEC-205 mAb with Lifastuzumab ScFv 531
HC Lifastuzumab with DEC-205 mAb ScFv 532
HC DEC-205 mAb with Sacituzumab ScFv 533
HC Sacituzumab with DEC-205 mAb ScFv 534
HC DEC-205 mAb with PR1A3 mAb ScFv 535
HC PR1A3 mAb with DEC-205 mAb ScFv 536
HC DEC-205 mAb with Humanized PR1A3 mAb ScFv 818
HC Humanized PR1A3 mAb with DEC-205 mAb ScFv 819
HC DEC-205 mAb with Humanized Ab2-3 mAb ScFv 836
HC Humanized Ab2-3 mAb with DEC-205 mAb ScFv 837
HC DEC-205 mAb with Claudiximab ScFv 537
HC Claudiximab with DEC-205 mAb ScFv 538
HC DEC-205 mAb with AMG595 mAb ScFv 539
HC AMG595 mAb with DEC-205 mAb ScFv 540
HC DEC-205 mAb with ABT806 mAb ScFv 541
HC ABT806 mAb with DEC-205 mAb ScFv 542
HC DEC-205 mAb with Sibrotuzumab ScFv 543
HC Sibrotuzumab with DEC-205 mAb ScFv 544
HC DEC-205 mAb with DS-8895a variant 1 mAb ScFv 545
HC DS-8895a variant 1 with DEC-205 mAb ScFv 546
HC DEC-205 mAb with DS-8895a variant 2 mAb ScFv 547
HC DS-8895a variant 2 mAb with DEC-205 mAb ScFv 548
HC DEC-205 mAb with Anti-EphA2 mAb ScFv 549
HC Anti-EphA2 mAb with DEC-205 mAb ScFv 550
HC DEC-205 mAb with Narnatumab mAb ScFv 551
HC Narnatumab with DEC-205 mAb ScFv 552
HC DEC-205 mAb with RG7841 mAb ScFv 553
HC RG7841 mAb with DEC-205 mAb ScFv 554
HC DEC-205 mAb with Farletuzumab ScFv 555
HC Farletuzumab with DEC-205 mAb ScFv 556
HC DEC-205 mAb with Mirvetuximab ScFv 557
HC Mirvetuximab with DEC-205 mAb ScFv 558
HC DEC-205 mAb with J591 variant 1 mAb ScFv 559
HC J591 variant 1 mAb with DEC-205 mAb ScFv 560
HC DEC-205 mAb with J591 variant 2 mAb ScFv 561
HC J591 variant 2 mAb with DEC-205 mAb ScFv 562
HC DEC-205 mAb with Rovalpituzumab mAb ScFv 563
HC Rovalpituzumab with DEC-205 mAb ScFv 564
HC DEC-205 mAb with PF-06647020 mAb ScFv 565
HC PF-06647020 mAb with DEC-205 mAb ScFv 566
HC DEC-205 mAb with anti-PTK7 mAb ScFv 567
HC anti-PTK7 mAb with DEC-205 mAb ScFv 568
HC DEC-205 mAb with Ladiratuzumab ScFv 569
HC Ladiratuzumab with DEC-205 mAb ScFv 570
HC DEC-205 mAb with Cirmtuzumab ScFv 571
HC Cirmtuzumab with DEC-205 mAb ScFv 572
HC DEC-205 mAb with anti-MAGE-A3 mAb ScFv 573
HC anti-MAGE-A3 mAb with DEC-205 mAb ScFv 574
HC DEC-205 mAb with anti-NY-ESO-1 mAb ScFv 575
HC anti-NY-ESO-1 mAb with DEC-205 mAb ScFv 576
HC DEC-205 mAb with Trastuzumab ScFv 686
HC Trastuzumab with DEC-205 mAb ScFv 687
111
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Fusion SEQ ID NO:
HC DEC205 mAb with Trastuzumab ScFv (LH,25mer) 797
TABLE 18. Exemplary Fusion Sequences ¨ CD40 fusions via the light chain
Fusion SEQ ID NO:
LC CD40 mAb containing Pertuzumab ScFv 766
LC Pertuzumab containing CD40 mAb ScFv 23
LC CD40 mAb containing Cetuximab ScFv 703
LC Cetuximab containing CD40 mAb ScFv 36
LC CD40 mAb containing Panitumumab ScFv 760
LC Panitumumab containing CD40 mAb ScFv 49
LC CD40 mAb containing Nimotuzumab ScFv 751
LC Nimotuzumab containing CD40 mAb ScFv 62
LC CD40 mAb containing Zalutumumab ScFv 802
LC Zalutumumab containing CD40 mAb ScFv 75
LC CD40 mAb containing Onartuzumab ScFv 757
LC Onartuzumab containing CD40 mAb ScFv 88
LC CD40 mAb containing Patritumab ScFv 763
LC Patritumab containing CD40 mAb ScFv 101
LC CD40 mAb containing Clivatuzumab ScFv 709
LC Clivatuzumab containing CD40 mAb ScFv 114
LC CD40 mAb containing Sofituzumab ScFv 790
LC Sofituzumab containing CD40 mAb ScFv 127
LC CD40 mAb containing Edrecolomab ScFv 718
LC Edrecolomab containing CD40 mAb ScFv 140
LC CD40 mAb containing Adecatumumab ScFv 694
LC Adecatumumab containing CD40 mAb ScFv 153
LC CD40 mAb containing Anetumab ScFv 700
LC Anetumab containing CD40 mAb ScFv 166
LC CD40 mAb containing huDS6 mAb ScFv 724
LC huDS6 mAb containing CD40 mAb ScFv 179
LC CD40 mAb containing Lifastuzumab ScFv 736
LC Lifastuzumab containing CD40 mAb ScFv 192
LC CD40 mAb containing Sacituzumab ScFv 781
LC Sacituzumab containing CD40 mAb ScFv 205
LC CD40 mAb containing PR1A3 mAb ScFv 772
LC PR1A3 mAb containing CD40 mAb ScFv 218
LC CD40 mAb containing Humanized PR1A3 mAb ScFv 820
LC Humanized PR1A3 mAb containing CD40 mAb ScFv 817
LC Humanized PR1A3 mAb containing CD40 mab scFv (LH) 844
LC CD40 mAb containing Humanized Ab2-3 mAb ScFv 838
LC Humanized Ab2-3 mAb containing CD40 mAb ScFv 835
LC Humanized Ab2-3 mAb containing CD40 mab scFv (LH) 842
LC CD40 mAb containing Claudiximab ScFv 727
LC Claudiximab containing CD40 mAb ScFv 231
LC CD40 mAb containing AMG595 mAb ScFv 697
LC AMG595 mAb containing CD40 mAb ScFv 270
LC CD40 mAb containing ABT806 mAb ScFv 691
LC ABT806 mAb containing CD40 mAb ScFv 283
LC CD40 mAb containing Sibrotuzumab ScFv 787
LC Sibrotuzumab containing CD40 mAb ScFv 296
LC CD40 mAb containing DS-8895a variant 1 mAb ScFv 712
LC DS-8895a variant 1 mAb containing CD40 mAb ScFv 309
LC CD40 mAb containing DS-8895a variant 2 mAb ScFv 715
LC DS-8895a variant 2 mAb containing CD40 mAb ScFv 322
LC CD40 mAb containing Anti-EphA2 mAb ScFv 742
LC Anti-EphA2 mAb containing CD40 mAb ScFv 335
LC CD40 mAb containing Narnatumab ScFv 748
112
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Fusion SEQ ID NO:
LC Narnatumab containing CD40 mAb ScFv 348
LC CD40 mAb containing RG7841 mAb ScFv 775
LC RG7841 mAb containing CD40 mAb ScFv 361
LC CD40 mAb containing Farletuzumab ScFv 721
LC Farletuzumab containing CD40 mAb ScFv 374
LC CD40 mAb containing Mirvetuximab ScFv 745
LC Mirvetuximab containing CD40 mAb ScFv 387
LC CD40 mAb containing J591 variant 1 mAb ScFv 730
LC J591 variant 1 mAb containing CD40 mAb ScFv 400
LC CD40 mAb containing J591 variant 2 mAb ScFv 733
LC J591 variant 2 mAb containing CD40 mAb ScFv 413
LC CD40 mAb containing Rovalpituzumab ScFv 778
LC Rovalpituzumab containing CD40 mAb ScFv 426
LC CD40 mAb containing PF-06647020 mAb ScFv 769
LC PF-06647020 mAb containing CD40 mAb ScFv 439
LC CD40 mAb containing anti-PTK7 mAb ScFv 688
LC anti-PTK7 mAb containing CD40 mAb ScFv 452
LC CD40 mAb containing Ladiratuzumab ScFv 784
LC Ladiratuzumab containing CD40 mAb ScFv 465
LC CD40 mAb containing Cirmtuzumab ScFv 706
LC Cirmtuzumab containing CD40 mAb ScFv 478
LC CD40 mAb containing anti-MAGE-A3 mAb ScFv 739
LC anti-MAGE-A3 mAb containing CD40 mAb ScFv 491
LC CD40 mAb containing anti-NY-ESO-1 mAb ScFv 754
LC anti-NY-ESO-1 mAb containing CD40 mAb ScFv 504
LC CD40 mAb containing Trastuzumab ScFv 793
LC Trastuzumab containing CD40 mAb ScFv 685
LC CD40 mAb containing Trastuzumab scFv (LH,25mer) 798
TABLE 19. Exemplary Fusion Sequences ¨ DEC-205 fusions via the light chain
Fusion SEQ ID NO:
LC DEC-205 mAb containing Pertuzumab ScFv 767
LC Pertuzumab containing DEC-205 mAb ScFv 768
LC DEC-205 mAb containing Cetuximab ScFv 704
LC Cetuximab containing DEC-205 mAb ScFv 705
LC DEC-205 mAb containing Panitumumab ScFv 761
LC Panitumumab containing DEC-205 mAb ScFv 762
LC DEC-205 mAb containing Nimotuzumab ScFv 752
LC Nimotuzumab containing DEC-205 mAb ScFv 753
LC DEC-205 mAb containing Zalutumumab ScFv 803
LC Zalutumumab containing DEC-205 mAb ScFv 804
LC DEC-205 mAb containing Onartuzumab ScFv 758
LC Onartuzumab containing DEC-205 mAb ScFv 759
LC DEC-205 mAb containing Patritumab ScFv 764
LC Patritumab containing DEC-205 mAb ScFv 765
LC DEC-205 mAb containing Clivatuzumab ScFv 710
LC Clivatuzumab containing DEC-205 mAb ScFv 711
LC DEC-205 mAb containing Sofituzumab ScFv 791
LC Sofituzumab containing DEC-205 mAb ScFv 792
LC DEC-205 mAb containing Edrecolomab ScFv 719
LC Edrecolomab containing DEC-205 mAb ScFv 720
LC DEC-205 mAb containing Adecatumumab ScFv 695
LC Adecatumumab containing DEC-205 mAb ScFv 696
LC DEC-205 mAb containing Anetumab ScFv 701
LC Anetumab containing DEC-205 mAb ScFv 702
LC DEC-205 mAb containing huDS6 mAb ScFv 725
LC huDS6 mAb containing DEC-205 mAb ScFv 726
113
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
Fusion SEQ ID NO:
LC DEC-205 mAb containing Lifastuzumab ScFv 737
LC Lifastuzumab containing DEC-205 mAb ScFv 738
LC DEC-205 mAb containing Sacituzumab ScFv 782
LC Sacituzumab containing DEC-205 mAb ScFv 783
LC DEC-205 mAb containing PR1A3 mAb ScFv 773
LC PR1A3 mAb containing DEC-205 mAb ScFv 774
LC DEC-205 mAb containing Humanized PR1A3 mAb ScFv 821
LC Humanized PR1A3 mAb containing DEC-205 mAb ScFv 822
LC DEC-205 mAb containing Humanized Ab2-3 mAb ScFv 839
LC Humanized Ab2-3 mAb containing DEC-205 mAb ScFv 840
LC DEC-205 mAb containing Claudiximab ScFv 728
LC Claudiximab containing DEC-205 mAb ScFv 729
LC DEC-205 mAb containing AMG595 mAb ScFv 698
LC AMG595 mAb containing DEC-205 mAb ScFv 699
LC DEC-205 mAb containing ABT806 mAb ScFv 692
LC ABT806 mAb containing DEC-205 mAb ScFv 693
LC DEC-205 mAb containing Sibrotuzumab ScFv 788
LC Sibrotuzumab containing DEC-205 mAb ScFv 789
LC DEC-205 mAb containing DS-8895a variant 1 mAb ScFv 713
LC DS-8895a variant 1 mAb containing DEC-205 mAb ScFv 714
LC DEC-205 mAb containing DS-8895a variant 2 mAb ScFv 716
LC DS-8895a variant 2 mAb containing DEC-205 mAb ScFv 717
LC DEC-205 mAb containing Anti-EphA2 mAb ScFv 743
LC Anti-EphA2 mAb containing DEC-205 mAb ScFv 744
LC DEC-205 mAb containing Narnatumab ScFv 749
LC Narnatumab containing DEC-205 mAb ScFv 750
LC DEC-205 mAb containing RG7841 mAb ScFv 776
LC RG7841 mAb containing DEC-205 mAb ScFv 777
LC DEC-205 mAb containing Farletuzumab ScFv 722
LC Farletuzumab containing DEC-205 mAb ScFv 723
LC DEC-205 mAb containing Mirvetuximab ScFv 746
LC Mirvetuximab containing DEC-205 mAb ScFv 747
LC DEC-205 mAb containing J591 variant 1 mAb ScFv 731
LC J591 variant 1 mAb containing DEC-205 mAb ScFv 732
LC DEC-205 mAb containing J591 variant 2 mAb ScFv 734
LC J591 variant 2 mAb containing DEC-205 mAb ScFv 735
LC DEC-205 mAb containing Rovalpituzumab ScFv 779
LC Rovalpituzumab containing DEC-205 mAb ScFv 780
LC DEC-205 mAb containing PF-06647020 mAb ScFv 770
LC PF-06647020 mAb containing DEC-205 mAb ScFv 771
LC DEC-205 mAb containing anti-PTK7 mAb ScFv 689
LC anti-PTK7 mAb containing DEC-205 mAb ScFv 690
LC DEC-205 mAb containing Ladiratuzumab ScFv 785
LC Ladiratuzumab containing DEC-205 mAb ScFv 786
LC DEC-205 mAb containing Cirmtuzumab ScFv 707
LC Cirmtuzumab containing DEC-205 mAb ScFv 708
LC DEC-205 mAb containing anti-MAGE-A3 mAb ScFv 740
LC anti-MAGE-A3 mAb containing DEC-205 mAb ScFv 741
LC DEC-205 mAb containing anti-NY-ESO-1 mAb ScFv 755
LC anti-NY-ESO-1 mAb containing DEC-205 mAb ScFv 756
LC DEC-205 mAb containing Trastuzumab ScFv 794
LC Trastuzumab containing DEC-205 mAb ScFv 795
LC DEC205 mAb containing Trastuzumab scFv (LH,25mer) 799
114
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
TABLE 20. Exemplary Fusion Sequences ¨ CD40 fusion with DEC205
Region SEQ ID NO:
HC DEC205 variant 1 mAb with CD40 mAb ScFv 243
HC CD40 mAb with DEC205 variant 1 mAb ScFv 242
LC DEC205 variant 1 mAb containing CD40 mAb scFv 244
LC CD40 mAb containing DEC205 variant lmAb scFv 800
HC DEC205 variant 2 mAb with CD40 mAb ScFv 256
HC CD40 mAb with DEC205 variant 2 mAb ScFv 255
LC DEC205 variant 2 mAb containing CD40 mAb scFv 257
LC CD40 mAb containing DEC205 variant 2 mAb scFv 801
[0271] In some aspects, an antibody construct comprising: a) a first binding
domain, wherein the
first binding domain specifically binds to a tumor antigen; b) a second
binding domain, wherein
the second binding domain specifically binds to an antigen on an antigen
presenting cell, wherein
the antigen is a molecule on the antigen presenting cell; and c) an Fc domain;
wherein the first
binding domain is attached to the Fc domain and the second binding domain is
attached to the Fc
domain or to a C-terminal end of a light chain of the first binding domain,
and wherein a Kd for
binding of the Fc domain to an Fc receptor in a presence of the first binding
domain and the
second binding domain is no greater than about 100 times a Kd for binding of
the Fc domain to
the Fc receptor in an absence of the second binding domain.
[0272] In some aspects, an antibody construct for use in inducing immune cell
activation
comprising: a) a first binding domain, wherein the first binding domain
specifically binds to a
tumor antigen; b) a second binding domain, wherein the second binding domain
specifically
binds to an antigen on an antigen presenting cell, wherein the antigen is a
molecule on the
antigen presenting cell; and c) an Fc domain; wherein the first binding domain
is attached to the
Fc domain and the second binding domain is attached to the Fc domain or to a C-
terminal end of
a light chain of the first binding domain, and wherein a Kd for binding of the
Fc domain to an Fc
receptor in a presence of the first binding domain and the second binding
domain is no greater
than about 100 times a Kd for binding of the Fc domain to the Fc receptor in
an absence of the
second binding domain; and wherein immune cell activation caused by the
antibody construct
upon binding to tumor antigen as measured by a cytokine release assay is
greater than immune
cell activation caused by the antibody construct in the absence of binding to
tumor antigen.
[0273] In some embodiments, the second binding domain is attached to the Fc
domain or the
light chain of the first binding domain: a) as an Fc domain-second binding
domain fusion peptide;
b) as a light chain-second binding domain fusion peptide; or c) by a
conjugation via a first linker.
In some embodiments, the Fc domain is attached to the first binding domain: a)
as an Fc domain-
first binding domain fusion peptide; or b) by conjugation via a second linker.
[0274] In some embodiments, the Fc domain is attached to both the first
binding domain and to
the second binding domain as a first binding domain-Fc domain-second binding
domain fusion
115
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
peptide. In some embodiments, the first binding domain is attached to both the
Fc domain and the
second binding domain as a first binding domain-second binding domain-Fc
domain fusion
peptide. In some embodiments, the first binding domain and the Fc domain
comprise an antibody
and the second binding domain comprises a single chain variable fragment
(scFv).
[0275] In some embodiments, the first binding domain has a set of variable
region CDR
sequences that comprises a set of variable region CDR sequences set forth in
TABLE 3 or
TABLE 4. In some embodiments, the second binding domain comprises a variable
domain
comprising a set of CDR sequences set forth in TABLE 11 or TABLE 12.
[0276] In some embodiments, the first binding domain comprises a variable
region comprising
VH and VL sequences at least 80% sequence identity to a pair of VH and VL
sequences set forth
in TABLE 5 or TABLE 6.
[0277] In some embodiments, the second binding domain comprises a variable
region having
VH and VL sequences having at least 80% sequence identity to a VH or VL
sequence set forth in
TABLE 13 or TABLE 14.
[0278] In some embodiments, the first binding domain comprises an amino acid
sequence having
at least 80% sequence identity to any sequence in TABLE 7 or TABLE 8. In some
embodiments,
the second binding domain comprises an amino acid sequence having at least 80%
sequence
identity to any sequence in TABLE 15 or TABLE 16.
[0279] In some embodiments, the second binding domain-Fc domain-first binding
domain fusion
peptide as described herein comprises an amino acid sequence having at least
80% sequence
identity to a sequence in TABLE 9, TABLE 10, or TABLE 17. In some embodiments,
the
second binding domain-first binding domain-Fc domain fusion peptide as
described herein
comprises an amino acid sequence having at least 80% sequence identity to a
sequence in
TABLE 18 or TABLE 19.
[0280] In some embodiments, the first binding domain comprises an amino acid
sequence having
at least 80% sequence identity to any sequence in TABLE 7 or TABLE 15.
[0281] In some embodiments, the antibody construct or conjugate induces
secretion of cytokines
by an immune cell as measured by a cytokine release assay. In some
embodiments, the cytokine
is IFN-y, IL-8, IL-12, IL-2, or a combination thereof. In some embodiments,
the antibody
construct or conjugate induces antigen presentation on a dendritic cell, B
cell, macrophage, or a
combination thereof.
Recombinant Bispecific Antibodies
[0282] In certain embodiments, recombinant antibodies are provided that are
"bispecific" that
possess the ability to specifically bind to two different targets through at
least two different
116
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
antigen binding domains (referred to as recombinant bispecific antibodies,
bispecific
recombinant antibodies or the like). These antibodies have a target antigen
binding domain and
an effector antigen binding domain. The target antigen binding domain
specifically binds to a
tumor associated antigen. The effector antigen binding domain specifically
binds to a molecule
present on an antigen presenting cell (APC).
[0283] The recombinant bispecific antibodies can exhibit more potent immune
activation when
both antigen binding domains are bound to their respective antigens. One
format for increasing
immune activation when both antigen binding domains are bound to their
respective antigens can
be accomplished by a recombinant bispecific antibody coupled to an Fc
comprising domain that
exhibits reduced affinity to an Fc receptor. Another format for achieving an
increased immune
activation when both antigen binding domains are bound to their respective
antigens can be
accomplished by using a binding domain with a low avidity for its antigen as
one of the antigen
binding domains in the recombinant bispecific antibody. One binding domain of
the bispecific
antibody can specifically bind to a tumor associated antigen and another
binding domain can
specifically bind to a molecule on the surface of an antigen presenting cell
(APC), such as a
macrophage or dendritic cell. Thus, the two binding domains cooperate to bring
APCs to
cancerous cells or tumors allowing the APC to initiate/propagate a cancer
cell/tumor specific
immune response through cytokine release, chemokine release, or presentation
of tumor
associated antigens to effector or helper T cells. Cytokine release can be
measured by a cytokine
release assay. Chemokine release can be measured by an ELISA immunoassay.
Presentation of
tumor associated antigens can be measured by a cross-presentation assay.
[0284] Therapy with recombinant monoclonal or bispecific antibodies can
generally be well
tolerated; however, antibodies directed to immune response stimulating
receptors on immune
cells can result in systemic toxic release of cytokines and other immune
modulators that can limit
their clinical use or dose, thereby limiting their effectiveness in generating
patient anti-tumor
responses. This immune activation can be especially non-beneficial when it
occurs systemically
in the absence of tumor antigens. The systemic agonism exhibited by antibodies
to many APC
receptors can depend upon high affinity binding to the APC antigen and higher
order cross-
linking of the APC receptors by clustering of the cell bound antibodies. Many
studies show this
can be mediated by Fc gamma receptor (FcyR) binding to the Fc domain of the
antibodies, and
cross-linking of different antibody molecules and their bound APC immune
stimulating receptors.
An additional complication of cross-linking by FcyR can be antibody dependent
cell mediated
cytotoxicity (ADCC) of the APCs resulting in lowered immune response to tumors
and
pathogens. ADCC can be attributed to the antibody Fc region which binds to
FcyRs on effector
117
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cells (e.g., NK cells). Two non-mutually exclusive solutions to the above can
be contemplated. In
one, elevating the threshold for FcyR binding can reduce excessive systemic
immune activation
and unwanted ADCC directed to APCs of antibody therapy. In the second, the
affinity of the
antibody for its APC target can be lowered so that effective agonistic binding
of antibody
molecules to APCs can be driven by avidity, preferentially found when the
bispecific antibody is
bound to its tumor antigen target. As described herein, the Fc comprising
domain of the
recombinant bispecific antibody can contain one or more mutations that can
reduce binding to an
FcyR. Alternatively, the Fc comprising domain can be derived from an IgG
subclass that can
bind to FcyRs with low affinity, for example IgG2. Fc receptors can be highly
expressed on
different antigen presenting cells such as dendritic cells, and their
engagement can lead to
activation of the immunostimulatory and antigen presenting function of these
cells. By reducing
binding of the Fc comprising domain to the FcyR, the threshold for APC
activation can be raised.
By raising the threshold for APC activation, the possibility of a damaging, or
non-beneficial,
immune/inflammatory response to healthy, non-cancerous tissue can be reduced.
Attenuating
activation by modifications made to the Fc comprising domain can result in
superior
bioavailability and lower side effects. Also described herein are recombinant
bispecific
antibodies with high affinity anti-tumor antigen binding and low affinity
immune receptor
binding such that APC activation can be increased when the recombinant
bispecific antibody is
bound to its tumor antigen. As a result, the antibodies of this disclosure
generally can have a
higher maximum tolerated dosage, and can be administered at levels higher than
therapeutic
antibodies not modified as described herein.
[0285] In some embodiments, a recombinant bispecific antibody comprises a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; an effector antigen binding domain, wherein the effector
antigen binding
domain specifically binds to a molecule present on an antigen presenting cell
and is not a
lipocalin mutein; and an Fc comprising domain; wherein the recombinant
bispecific antibody
induces more potent immune activation when the recombinant bispecific antibody
is bound to the
tumor associated antigen and to the molecule on the antigen presenting cell as
compared to when
the recombinant bispecific antibody is bound to the molecule on the antigen
presenting cell but
not to the tumor associated antigen. In some aspects, immune activation by the
recombinant
bispecific antibody when bound to the tumor associated antigen is at least two
times, five times,
or ten times greater than immune activation by the recombinant bispecific
antibody when the
recombinant bispecific antibody is not bound to the tumor associated antigen.
118
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0286] In some embodiments, immune activation by the recombinant bispecific
antibody is
greater in the presence of cells having cell surface tumor associated antigen
and antigen
presenting cells having cell surface molecule as compared to immune activation
in the absence of
cells having cell surface tumor associated antigen. In some embodiments,
immune activation by
the recombinant bispecific antibody is greater in the presence of cells having
cell surface tumor
associated antigen and antigen presenting cells having cell surface molecule
as compared to
immune activation in the absence of cells having cell surface tumor associated
antigen but in the
presence of the antigen presenting cells.
[0287] In some embodiments, a recombinant bispecific antibody comprises a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; an effector antigen binding domain, wherein the effector
antigen binding
domain specifically binds to a molecule present on an antigen presenting cell
and is not a
lipocalin mutein; and an Fc comprising domain; wherein when administered at
the minimum
anticipated biological effect level of the recombinant bispecific antibody,
the biological effect of
the recombinant bispecific antibody is increased when the recombinant
bispecific antibody is
bound to the tumor associated antigen as compared to the biological effect of
the recombinant
bispecific antibody when the recombinant bispecific antibody is not bound to
the tumor
associated antigen, but is bound to the molecule on the antigen presenting
cell. In some
embodiments, the biological effect is immune activation. In some aspects,
biological effect of the
recombinant bispecific antibody when the recombinant bispecific antibody is
bound to the tumor
associated antigen is at least two times, five times, or ten times greater
than the biological effect
of the recombinant bispecific antibody when the recombinant bispecific
antibody is not bound to
the tumor associated antigen, but is bound to the molecule on the antigen
presenting cell.
[0288] In further aspects, an increase in biological effect (e.g., immune
activation) is an increase
one or more of: a secretion of one or more cytokines, a secretion of one or
more chemokines, an
expression level of one or more cell surface proteins associated with immune
stimulation, and an
activity of one or more immune cell functions. In still further aspects, the
activity of one or more
immune cell functions comprises antibody-dependent cell-mediated cytotoxicity,
antibody
dependent cellular phagocytosis, or antigen cross-presentation. In some
aspects, the increase in
immune activation is two times, three times, five times, or ten times greater
than immune
activation by the recombinant bispecific antibody when the recombinant
bispecific antibody is
not bound to the tumor associated antigen, but is bound to the molecule on the
antigen presenting
cell. In further aspects, the recombinant bispecific antibody induces tumor-
cell directed antibody-
dependent cell-mediated cytotoxicity.
119
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0289] In some aspects, a binding affinity of the effector antigen binding
domain of the
recombinant bispecific antibody to the molecule present on the antigen
presenting cell is
decreased compared to a binding affinity of the effector antigen binding
domain of an antibody
that lacks the target antigen binding domain, and wherein the binding affinity
of the effector
antigen binding domain of the recombinant bispecific antibody to the molecule
present on the
antigen presenting cell in a recombinant form is similar to the binding
affinity of the effector
antigen binding domain of an antibody that lacks the target antigen binding
domain. In further
aspects, a Kd of the binding affinity of the effector antigen binding domain
of the recombinant
bispecific antibody to the molecule present on the antigen presenting cell is
increased by two
times, five times, ten times, fifty times, or one-hundred times compared to
the binding affinity of
the effector antigen binding domain of an antibody that lacks the target
antigen binding domain.
In additional aspects, a Kd for binding of the effector antigen binding domain
to the molecule
present on the antigen presenting cell is less than 20 nM, less than 100 nM,
or less than 500 nM.
[0290] In some aspects, the Fc comprising domain comprises one or more amino
acid
substitutions that result in an increase in affinity to one or more Fey
receptors as compared to a
wild-type Fc comprising domain. In other aspects, the Fc comprising domain
comprises one or
more amino acid substitutions that result in a decrease in affinity to one or
more Fey receptors as
compared to a wild-type Fc comprising domain. In some aspects, the Fc
comprising domain
comprises one or more amino acid substitutions that result in a loss of
binding to one or more Fey
receptors as compared to a wild-type Fc comprising domain.
[0291] These recombinant bispecific antibodies can be designed in many
different configurations.
In certain embodiments, the bispecific antibodies comprise two different
antigen binding
domains and an Fc comprising domain. In certain embodiments, the Fc comprising
domain
comprises an immunoglobulin constant region or portion thereof. The Fc
comprising domain
comprises at least one mutation that reduces the affinity of the Fc domain for
an Fc receptor. In
certain aspects, the Fc comprising domain comprises at least 2, 3, 4, 5, 6, 7,
8,9, 10 or more
mutations that reduce the affinity of the comprising domain for an Fc
receptor. In certain
embodiments, the Fc comprising domain comprises the Fc region of the IgG1
isotype subclass.
In certain embodiments, the Fc comprising domain comprises the Fc region of
the IgG2 isotype
subclass. Alternatively, the Fc comprising domain comprises the Fc region of
the IgG2A isotype
subclass. The target antigen binding domain is arranged in any configuration
that exhibits
specific binding to a desired molecule and comprise at least one CDR sequence.
For example, in
certain embodiments, a binding domain comprises a Fab, an scFv, a heavy chain
variable binding
region capable of specific binding without a corresponding light chain (e.g.,
a camelid heavy
120
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
chain antibody), or a nanobody. The effector antigen binding domain is
arranged in any
configuration that exhibits specific binding to a desired molecule and
comprises at least one CDR
sequence or polypeptide that specifically binds to a molecule present on the
surface of an APC.
Antigen binding domains can either be attached or coupled to the Fc comprising
domain. For
example, an antigen binding domain is attached to the Fc comprising domain
when it is encoded
by the same polypeptide (e.g., a fusion protein of two different polypeptides
connected by
peptide bonds between two amino acids). This attachment can be effected by
using peptide
synthesis techniques or production in a cell based system. The antigen binding
domains can also
be coupled to the Fc comprising domain via a covalent linkage that is not a
peptide bond, for
example, via a polyethylene glycol (PEG) linker.
[0292] FIGURE 10 depicts at least three examples of non-limiting embodiments
of the
recombinant bispecific antibodies. In embodiment I, a target antigen binding
domain comprises a
light chain variable and constant region 101 and a heavy chain variable and
constant region 102.
The heavy chain variable and constant region, either comprises an Fc region
itself, or is attached
to a polypeptide comprising an Fc region 103, which is in turn attached to an
effector antigen
binding domain comprising an scFv which comprises a heavy chain variable
region 104 and a
light chain variable region 105. In embodiment I the recombinant bispecific
antibody is formed
from two separate polypeptides. A single polypeptide encodes the heavy chain
variable region
102, the Fc region 103, and the scFv 104 and 105, while a second polypeptide
encodes the light
chain variable region and constant region 101. The second polypeptide is
covalently coupled to
the first polypeptide by a disulfide linkage. In this embodiment, the target
antigen binding
domain approximates a Fab fragment. Each polypeptide comprises a leader
sequence (not shown)
that directs the polypeptide to the endoplasmic reticulum and the secretory
pathway. The leader
sequence is ultimately cleaved off of the polypeptides and the secreted
molecule lacks the leader
sequence. In a certain embodiment, the target antigen binding domain, the Fc
region, and the
effector antigen binding domain are attached via linkers that allow freedom of
movement of the
binding domains and prevent steric hindrance by the Fc region. The linkers can
be any suitable
length greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In
certain embodiments, the
linkers are resistant to proteases. In embodiment I, while the target antigen
binding domain is
depicted at the N-terminus and the effector antigen binding domain scFv at the
C-terminus, the
orientation can be reversed with the effector antigen binding domain at the N-
terminus and the
target antigen binding domain at the C-terminus. The target antigen binding
domain specifically
binds to a tumor associated antigen; the effector antigen binding domain
specifically binds to a
molecule present on an antigen presenting cell. It is also envisioned that the
Fab fragment can be
121
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
expressed separately from the Fc region-scFv 103-105 and coupled by chemical
means through
the use of a linker.
[0293] In alternative embodiment II, the recombinant bispecific antibody
comprises a single
polypeptide with two different antigen binding domains that comprise scFvs. A
first scFv
comprises a light chain variable region 106 attached to a heavy chain variable
region 107, which
is in turn attached to a polypeptide comprising an Fc region 108, which is
attached to a second
scFv comprising a heavy chain variable region 109 and a light chain variable
region 110. In
certain embodiments, the target antigen binding domain specifically binds to a
tumor associated
antigen, while the effector antigen binding domain specifically binds to a
molecule present on an
antigen presenting cell.
[0294] In alternative embodiment III, the recombinant bispecific antibody
comprises two
different Fab fragments (111 and 112; 114 and 115) that are coupled or
attached to a polypeptide
comprising an Fc region 113.
[0295] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen binding
domain, wherein the target antigen binding domain specifically binds to a
tumor associated
antigen; b) an effector antigen binding domain, wherein the effector antigen
binding domain
specifically binds to an antigen on an antigen presenting cell and wherein the
antigen is a
molecule on the antigen presenting cell; c) an Fc comprising domain; and d) an
immune-
stimulatory compound attached to the recombinant bispecific antibody by a
linker; wherein the
recombinant bispecific antibody induces greater immune cell activation when
the recombinant
bispecific antibody is bound to the tumor associated antigen and to the
antigen on the antigen
presenting cell as compared to when the recombinant bispecific antibody is
bound to the antigen
on the antigen presenting cell but not to the tumor associated antigen.
[0296] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen binding
domain, wherein the target antigen binding domain specifically binds to a
tumor associated
antigen; b) an effector antigen binding domain, wherein the effector antigen
binding domain
specifically binds to an antigen on an antigen presenting cell and is an
antibody antigen binding
domain, wherein the antigen is a molecule on the antigen presenting cell; and
c) a domain
comprising an Fc region;wherein the recombinant bispecific antibody induces
greater immune
cell activation when the recombinant bispecific antibody is bound to the tumor
associated antigen
and to the antigen on the antigen presenting cell as compared to when the
recombinant bispecific
antibody is bound to the antigen on the antigen presenting cell but not to the
tumor associated
antigen.
122
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0297] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen binding
domain, wherein the target antigen binding domain specifically binds to a
tumor associated
antigen; b) an effector antigen binding domain, wherein the effector antigen
binding domain
specifically binds to an antigen on an antigen presenting cell and is an
antibody antigen binding
domain, wherein the antigen is a molecule on the antigen presenting cell; and
c) a domain
comprising an Fc region; wherein the recombinant bispecific antibody induces
greater immune
cell activation in the presence of cells having cell surface tumor associated
antigen and antigen
presenting cells having cell surface antigen as compared to immune cell
activation in the absence
of cells having cell surface tumor associated antigen.
[0298] In some aspects, a recombinant bispecific antibody, comprising: a) a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; b) an effector antigen binding domain, wherein the
effector antigen binding
domain specifically binds to an antigen on an antigen presenting cell and
wherein the antigen is a
molecule on the antigen presenting cell; and c) an Fc comprising domain; and
d) an immune-
stimulatory compound attached to the recombinant bispecific antibody by a
linker; wherein the
recombinant bispecific antibody induces greater immune cell activation in the
presence of cells
having cell surface tumor associated antigen and antigen presenting cells
having cell surface
antigen as compared to immune cell activation in the absence of cells having
cell surface tumor
associated antigen.
[0299] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen binding
domain, wherein the target antigen binding domain specifically binds to a
tumor associated
antigen; b) an effector antigen binding domain, wherein the effector antigen
binding domain
specifically binds to an antigen on an antigen presenting cell and wherein the
antigen is a
molecule on the antigen presenting cell; c) an Fc comprising domain; and d) an
immune-
stimulatory compound attached to the recombinant bispecific antibody by a
linker; wherein the
recombinant bispecific antibody induces greater immune cell activation when
the recombinant
bispecific antibody is bound to the tumor associated antigen and to the
antigen on the antigen
presenting cell as compared to when the recombinant bispecific antibody is
bound to the antigen
on the antigen presenting cell but not to the tumor associated antigen.
[0300] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen binding
domain, wherein the target antigen binding domain specifically binds to a
tumor associated
antigen; b) an effector antigen binding domain, wherein the effector antigen
binding domain
specifically binds to an antigen on an antigen presenting cell and is an
antibody antigen binding
domain, wherein the antigen is a molecule on the antigen presenting cell; and
c) a domain
123
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprising an Fc region;wherein the recombinant bispecific antibody induces
greater immune
cell activation when the recombinant bispecific antibody is bound to the tumor
associated antigen
and to the antigen on the antigen presenting cell as compared to when the
recombinant bispecific
antibody is bound to the antigen on the antigen presenting cell but not to the
tumor associated
antigen.
[0301] In some aspects, a recombinant bispecific antibody, comprises: a) a
target antigen binding
domain, wherein the target antigen binding domain specifically binds to a
tumor associated
antigen; b) an effector antigen binding domain, wherein the effector antigen
binding domain
specifically binds to an antigen on an antigen presenting cell and is an
antibody antigen binding
domain, wherein the antigen is a molecule on the antigen presenting cell; and
c) a domain
comprising an Fc region; wherein the recombinant bispecific antibody induces
greater immune
cell activation in the presence of cells having cell surface tumor associated
antigen and antigen
presenting cells having cell surface antigen as compared to immune cell
activation in the absence
of cells having cell surface tumor associated antigen.
[0302] In some aspects, a recombinant bispecific antibody, comprising: a) a
target antigen
binding domain, wherein the target antigen binding domain specifically binds
to a tumor
associated antigen; b) an effector antigen binding domain, wherein the
effector antigen binding
domain specifically binds to an antigen on an antigen presenting cell and
wherein the antigen is a
molecule on the antigen presenting cell; and c) an Fc comprising domain; and
d) an immune-
stimulatory compound attached to the recombinant bispecific antibody by a
linker; wherein the
recombinant bispecific antibody induces greater immune cell activation in the
presence of cells
having cell surface tumor associated antigen and antigen presenting cells
having cell surface
antigen as compared to immune cell activation in the absence of cells having
cell surface tumor
associated antigen.
[0303] In some embodiments, the Fc comprising domain is linked C-terminal to
the target
antigen binding domain and N-terminal to the effector antigen binding domain.
In some
embodiments, the linker links the immune-stimulatory compound to the Fc
comprising domain.
[0304] In some embodiments, the immune cell activation is measured by a
cytokine release assay.
[0305] In some embodiments, the immune cell activation by the recombinant
bispecific antibody
when the recombinant bispecific antibody is bound to the tumor associated
antigen and to the
antigen on the antigen presenting cell is at least two times, five times, or
ten times greater than
immune activation by the recombinant bispecific antibody when the recombinant
bispecific
antibody is bound to the antigen on the antigen presenting cell but not to the
tumor associated
antigen as measured by the cytokine release assay.
124
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0306] In some embodiments, the immune cell activation by the recombinant
bispecific antibody
in the presence of cells having cell surface tumor associated antigen and
antigen presenting cells
having cell surface antigen is at least two times, five times, or ten times
greater than immune cell
activation by the recombinant bispecific antibody in the absence of the cells
having cell surface
tumor associated antigen as measured by the cytokine release assay.
[0307] In some embodiments, the immune cell activation comprises an increase
in one or more
of: a) a secretion of one or more cytokines as measured by the cytokine
release assay, b) a
secretion of one or more chemokines as measured by an ELISA immunoassay, c) an
expression
level of one or more cell surface proteins associated with immune stimulation
as measured by
FACS, and d) an activity of one or more immune cell functions.
[0308] In some embodiments, the activity of one or more immune cell functions
comprises
antibody-dependent cell-mediated cytotoxicity as measured by an ADCC assay,
antibody
dependent cellular phagocytosis as measured by an ADCP assay, or antigen cross-
presentation as
measured by a cross-presentation assay.
[0309] In some embodiments, the recombinant bispecific antibody induces tumor-
cell directed
antibody-dependent cell-mediated cytotoxicity.
[0310] In some aspects, method of making a recombinant bispecific antibody
comprises: a)
producing an antibody construct comprising: i) a target antigen binding
domain, wherein the
target antigen binding domain specifically binds to a tumor associated
antigen; ii) an effector
antigen binding domain, wherein the effector antigen binding domain
specifically binds to an
antigen on an antigen presenting cell and the antigen is a molecule on the
antigen presenting cell,
wherein the antigen is a molecule on the antigen presenting cell; iii) an Fc
comprising domain;
and b) linking an immune-stimulatory compound to the antibody construct,
wherein the
recombinant bispecific antibody induces greater immune cell activation when
the recombinant
bispecific antibody is bound to the tumor associated antigen and to the
antigen on the antigen
presenting cell as compared to when the recombinant bispecific antibody is
bound to the antigen
on the antigen presenting cell but not to the tumor associated antigen.
Constant Regions and Fc Regions
[0311] The recombinant bispecific antibodies described herein comprise a
constant region, or
portion thereof, in addition to a variable region (or CDR sequences derived
from a variable
region). The heavy chain constant region (CH) comprises three or four domains
abbreviated CH1,
CH2, CH3, and CH4, depending on the isotype of the constant region. The
domains are located
at the C-terminal end of the full heavy chain polypeptide, C-terminal to the
variable region. The
light chain constant region (CL) is much smaller that the CH and is located at
the C-terminal end
125
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
of the full light chain polypeptide, C-terminal to the variable region. In
certain embodiments, the
recombinant bispecific antibodies herein comprise a constant region lacking a
CH4 region. The
constant region is highly conserved and comprises different isotypes, that are
associated with
slightly different functions and properties. In certain embodiments, the
constant region is not
required for antibody binding to a target antigen. In certain embodiments, the
constant regions of
the recombinant bispecific antibodies, both heavy and light chains are not
required for antibody
binding to a target antigen. In certain embodiments, the recombinant
bispecific antibodies lack
one or more of a light chain constant region, heavy chain constant region, or
both. Most
monoclonal antibodies are of an IgG isotype; which is further divided into
four subclasses IgGl,
IgG2, IgG3, and IgG4. In certain embodiments, the recombinant bispecific
antibodies comprise
any IgG subclass. In certain embodiments, the IgG subclass comprises IgGl. In
certain
embodiments, the IgG subclass comprises IgG2. In certain embodiments, the IgG
subclass
comprises IgG3. In certain embodiments, the IgG subclass comprises IgG4.
[0312] The recombinant bispecific antibodies described herein comprise an Fc
comprising
domain in addition to target and effector antigen binding domains. Natural
antibodies comprise a
fragment crystallizable region (Fc region) that is responsible for binding to
complement and Fc
receptors. The Fc region comprises the CH2, CH3, and CH4 regions of the
antibody molecule,
and is responsible for activating complement and antibody dependent cell
cytotoxicity (ADCC).
Much of the variability in Fc function can be attributed to the CH2 region.
The Fc region also
contributes to an antibody's serum half-life. IgG isotype subclass Fc regions
exhibit varying
affinity for Fc receptors. IgG1 and IgG3 exhibit high affinity binding to all
Fc receptors, both
with respect to affinity and amount of different Fc receptors. IgG2, however,
exhibits the lowest
binding affinity toward various Fc receptors, with IgG4 showing an
intermediate binding affinity.
In certain embodiments, the Fc region is attached to one or both of the
antigen binding domains.
In certain embodiments, the recombinant bispecific antibodies comprise an Fc
region derived
from the IgG2 isotype subclass.
[0313] There are several different Fc receptors with varying affinity for the
antibody Fc region,
ability to activate signaling though the Fc receptor, and expression on immune
cells. For example,
FcyRI possess a very high affinity for IgG (KD of approximately lx10-8 M).
[0314] The Fc comprising domain of the recombinant bispecific antibodies
comprises one or
more, two or more, three or more, or four or more amino acid substitutions
that decrease binding
of the antibody to an Fc receptor. In certain embodiments, the Fc receptor
comprises FcyRI
(CD64), FcyRIIA (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD16b), or any combination
thereof.
In certain embodiments, the Fc comprising domain of the recombinant bispecific
antibodies
126
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprise one or more amino acid substitutions that increase the serum half-
life of the antibody.
In certain embodiments, the one or more amino acid substitutions that increase
the serum half-
life of the antibody increase affinity of the antibody to the neonatal Fc
receptor (FcRn). In order
to decrease binding affinity of an Fc region to Fc receptor the Fc comprising
domain may
comprise one or more mutations that has the effect of reducing the affinity of
the Fc region to an
Fc receptor. In certain embodiments, the one or more mutations comprise any
one or more of
IgG1 heavy chain mutations corresponding to E233P, L234V, L234A, L235A, L235E,
AG236,
G237A, E318A, K320A, K322A, A327G, A330S, or P33 1S according to the EU index
of Kabat
numbering.
[0315] The Fc comprising domain of the recombinant bispecific antibodies
comprises one, two,
three, four or more amino acid substitutions that decrease binding of the
antibody to an Fc
receptor. The one, two, three, four or more amino acid substitutions decrease
binding by at least
two fold, three fold, four fold, five fold or ten fold. In certain
embodiments, the one, two, three,
four or more amino acid substitutions completely abolish binding to an Fc
receptor (Fc null). In
certain embodiments, the one, two, three, four or more amino acid
substitutions are differences
compared to any one of SEQ ID NO: 1314, SEQ ID NO: 1315, SEQ ID NO: 1316, or
SEQ ID
NO: 1317. In certain embodiments, the one, two, three, four or more amino acid
substitutions are
differences compared to any one of SEQ ID NO: 1314, SEQ ID NO: 1315, SEQ ID
NO: 1316, or
SEQ ID NO: 1317. In certain embodiments, one, two, three, four or more amino
acid
substitutions are differences compared to any one of SEQ ID NO: 1319, SEQ ID
NO: 1320, SEQ
ID NO: 1321, or SEQ ID NO: 1322. In certain embodiments, one, two, three, four
or more amino
acid substitutions are differences compared to any one of SEQ ID NO: 1319, SEQ
ID NO: 1320,
SEQ ID NO: 1321, or SEQ ID NO: 1322. In certain embodiments, the Fc comprising
domain
comprises an amino sequence at least 80%, 90%, 95%, 98%, 99%, or 100%
identical to the
amino acid sequence set forth in SEQ ID NO: 1313. In certain embodiments, the
Fc comprising
domain comprises an amino sequence at least 80%, 90%, 95%, 98%, 99%, or 100%
identical to
the amino acid sequence set forth in SEQ ID NO: 1318.
[0316] Alternatively, an amino acid change in an Fc comprising domain of the
recombinant
bispecific antibodies can allow the recombinant bispecific antibody to bind to
at least one Fc
receptor with greater affinity compared to a wild-type Fc region. An Fc
comprising domain can
comprise an amino acid sequence having at least one, two, three, four, five,
six, seven, eight, nine
or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10
modifications of the amino
acid sequence relative to the natural or original amino acid sequence. An Fc
comprising domain
can be an Fc region of an anti-CD40 antibody.
127
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0317] The Fc comprising domain of the recombinant bispecific antibody can
comprise a
sequence of the IgG1 isoform that has been modified from the wild-type IgG1
sequence. A
modification can comprise a substitution at more than one amino acid residue
such as at 5
different amino acid residues including L235V/F243L/R292P/Y300L/P396L
(IgG1VLPLL). The
numbering of amino acids residues described herein is according to the EU
index as in Kabat.
The 5 amino acid residues can be located in a portion of a recombinant
bispecific antibody
sequence which can encode an Fc comprising domain of the recombinant
bispecific antibody and
in particular, can be located in portions of the Fc region that can bind to Fc
receptors (i.e., the Fc
domain). A modification can comprise a substitution at more than one amino
acid residue such as
at 2 different amino acid residues including S239D/I332E (IgG1DE) according to
the EU index
of Kabat numbering. The 2 amino acid residues can be located in a portion of a
recombinant
bispecific antibody sequence which encodes an Fc comprising domain of the
recombinant
bispecific antibody and in particular, are located in portions of the Fc
region that can bind to Fc
receptors (i.e., the Fc domain). A modification can comprise a substitution at
more than one
amino acid residue such as at 3 different amino acid residues including
S298A/E333A/K334A
(IgGlAAA) according to the EU index of Kabat numbering. The 3 amino acid
residues can be
located in a portion of a recombinant bispecific antibody sequence which can
encode an Fc
comprising domain of the recombinant bispecific antibody and in particular,
can be located in
portions of the Fc region that can bind Fc receptors (i.e., the Fc domain).
[0318] The Fc comprising domain can be from a monoclonal anti-CD40 human
antibody
comprising a sequence of the IgG1 isoform that has been modified from the wild-
type IgG1
sequence. A modification can comprise a substitution at more than one amino
acid residue such
as at 5 different amino acid residues including L235V/F243L/R292P/Y300L/P396L
(SBT-040-
G1VLPLL). The numbering of amino acids residues described herein is according
to the EU
index as in Kabat. The 5 amino acid residues can be located in a portion of an
antibody sequence
which can encode an Fc region of the antibody and in particular, can be
located in portions of the
Fc region that can bind to Fc receptors (i.e., the Fc domain). A modification
can comprise a
substitution at more than one amino acid residue such as at 2 different amino
acid residues
including S239D/I332E (SBT-040-G1DE) according to the EU index of Kabat
numbering. The 2
amino acid residues can be located in a portion of an antibody sequence which
encodes an Fc
region of the antibody and in particular, are located in portions of the Fc
region that can bind to
Fc receptors (i.e., the Fc domain). A modification can comprise a substitution
at more than one
amino acid residue such as at 3 different amino acid residues including
S298A/E333A/K334A
(SBT-040-G1AAA) according to the EU index of Kabat numbering. The 3 amino acid
residues
128
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
can be located in a portion of an antibody sequence which can encode an Fc
region of the
antibody and in particular, can be located in portions of the Fc region that
can bind to Fc
receptors (i.e., the Fc domain).
[0319] Binding of Fc receptors to an Fc comprising domain can be affected by
amino acid
substitutions. For example, a recombinant bispecific antibody can comprise an
amino acid
sequence of a heavy chain of human anti-CD40 monoclonal antibody with
modifications to a
wild-type IgG1 Fc domain (L235V/F243L/R292P/Y300L/P396L). Binding of some Fc
receptors
to this Fc comprising domain with the L235V/F243L/R292P/Y300L/P396L amino acid
modifications can be enhanced compared to wild-type as result of the
L235V/F243L/R292P/Y300L/P396L amino acid modifications. The recombinant
bispecific
antibody can comprise an amino acid sequence of a heavy chain of human anti-
CD40
monoclonal antibody with modifications to a wild-type IgG1 Fc domain
(S239D/I332E). Binding
of Fc receptors to the Fc comprising domain of the recombinant bispecific
antibody can be
enhanced compared to wild-type as a result of the S239D/I332E amino acid
modification. The
recombinant bispecific antibody can comprise an amino acid sequence of a heavy
chain of a
human anti-CD40 monoclonal antibody with modifications to a wild-type IgG1 Fc
domain
(S298A/E333A/K334A). Binding of Fc receptors to a Fc comprising domain of a
recombinant
bispecific antibody can be enhanced compared to wild-type as a result of the
S298A/E333A/K334A amino acid modification.
[0320] In some embodiments, the Fc comprising domain has one or more amino
acid
substitutions that decrease the binding affinity to one or more Fey receptors
as compared to a
wild-type Fc comprising domain.
[0321] In some embodiments, the Fc comprising domain is linked to the target
antigen binding
domain and to the effector antigen binding domain.
[0322] In some embodiments, the Fc comprising domain comprises one or more
amino acid
substitutions that reduce the affinity of the Fc comprising domain to an Fc
receptor compared to
the affinity of a reference Fc comprising domain to the Fc receptor in the
absence of the one or
more amino acid substitutions.
[0323] In some embodiments, reference Fc comprising domain is selected from
the group
consisting of an Fc comprising domain having the amino acid sequence of SEQ ID
NO: 1314,
SEQ ID NO: 1315, SEQ ID NO: 1316, and SEQ ID NO: 1317.
[0324] In some embodiments, reference Fc comprising domain comprises an amino
acid
sequence selected from the group consisting of SEQ ID NO: 1319, SEQ ID NO:
1320, SEQ ID
NO: 1321, and SEQ ID NO: 1322.
129
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0325] In some embodiments, the Fc comprising domain comprises a human IgGi Fc
Region. In
some embodiments, the one or more amino acid substitutions comprise L234A,
L235A, G237A,
and K322A, according to the EU index of Kabat. In some embodiments, the one or
more amino
acid substitutions comprise E233P, L234V, L235A, AG236, A327G, A330S, and P33
1S,
according to the EU index of Kabat. In some embodiments, the Fc comprising
domain comprises
a human IgG2 Fc Region.
[0326] In some embodiments, the one or more amino acid substitutions comprises
K322A,
according to the EU index of Kabat.
[0327] In some embodiments, the Fc comprising domain comprises a human IgG2a
Fc Region In
some embodiments, the one or more amino acid substitutions comprises L235E,
E318A, K320A,
K322A, according to the EU index of Kabat.
[0328] In some embodiments, the Fc comprising domain is an Fc null. In some
embodiments, the
Fc comprising domain has the amino acid sequence of SEQ ID NO: 1313.
[0329] In some embodiments, the Fc comprising domain comprises the amino acid
sequence of
SEQ ID NO: 1318.
[0330] In some embodiments, the Fc comprising domain is linked C-terminal to
the target
antigen binding domain and has the amino acid sequence of SEQ ID NO: 1311.
Antigen Binding Domains
[0331] In certain embodiments, the recombinant bispecific antibodies comprise
a target and
effector antigen binding domain. Each antigen binding domain comprises one or
more
complementarity determining regions (CDRs). A CDR is a part of an
immunoglobulin (antibody)
variable region that is primarily responsible for the antigen binding
specificity of the antibody.
CDR regions are highly variable from one antibody to the next, even when the
antibody
specifically binds the same target or epitope. A heavy chain variable region
comprises three CDR
regions, abbreviated HCDR1, HCDR2, and HCDR3; and a light chain variable
region comprises
three CDR regions, abbreviated LCDR1, LCDR2, and LCDR3. These CDR regions are
ordered
consecutively in the variable region with the CDR1 being the most N-terminal
and the CDR3
being the most C-terminal. Interspersed between the CDRs are framework regions
which
contribute to the structure and display much less variability than the CDR
regions. A heavy chain
variable region comprises four framework regions, abbreviated HFR1, HFR2,
HFR3, and HFR4;
and a light chain variable region comprises four framework regions,
abbreviated LFR1, LFR2,
LFR3, and LFR4. Complete full sized bivalent antibodies comprising two heavy
and light chains
will comprise: 12 CDRs, with three unique heavy chain CDRs and three unique
light chain CDRs;
16 FR regions, with four unique heavy chain FR regions and four unique light
chain FR regions.
130
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
In certain embodiments, the antibodies described herein minimally comprise
three heavy chain
CDRs. In certain embodiments, the antibodies described herein minimally
comprise three light
chain CDRs. In certain embodiments, the antibodies described herein minimally
comprise three
heavy chain CDRs and three light chain CDRs. The CDRs may be expressed as an
scFv or a
traditional heavy chain and light chain pair. CDRs are identified from
sequences using different
numbering systems such as the Kabat or the IMGT numbering systems. In certain
embodiments,
the antibodies described herein comprise variable regions of non-human origin.
In certain
embodiments, the antibodies described herein comprise CDRs of non-human
origin. In certain
embodiments, the antibodies described herein comprise variable regions of
mouse origin. In
certain embodiments, the antibodies described herein comprise CDRs of mouse
origin.
[0332] In some embodiments, the tumor associated antigen is an antigen
selected from the group
consisting of CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, HLD-
DR,
carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-binding
protein,
A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2,
Leg, CA-
125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, a
metalloproteinase,
endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV
E7, EGFRvIII,
Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, MelanA/MART1, Ras mutant, gp100,
p53
mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a Sarcoma
translocation breakpoint
fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen
receptor,
cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN),
PSCA,
MAGE Al, MAGE-A3, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-
AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, 0Y-TES1, Sperm
protein
17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2,
MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EPCAM, CA6, NAPI2B, TROP2,
CLDN18.2, fibroblast activation protein (FAP), RON, LY6E, FRA, DLL3, PTK7,
LIV1, ROR1,
Fos-related antigen 1, VEGFR, endoglin, PD-L1, CD204, CD206, CD301, VTCN1, and
VISTA.
In some embodiments, the tumor associated antigen is Her2/neu or p185HER2.
Target Antigen Binding Domain
[0333] The recombinant bispecific antibodies comprise a target antigen binding
domain that
specifically binds to a tumor associated antigen. As described herein, a
"tumor associated antigen"
refers to a molecular marker that is can be expressed by a neoplastic tumor
cell and/or within a
tumor microenvironment. For example, a tumor associated antigen can be an
antigen expressed
on a cell associated with a tumor, such as a neoplastic cell, stromal cell,
endothelial cell,
fibroblast, or tumor-infiltrating immune cell. For example, the tumor
associated antigen
131
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Her2/Neu is overexpressed by certain types of breast and ovarian cancer. A
tumor antigen may
also be ectopically expressed by a tumor and contribute to deregulation of the
cell cycle, reduced
apoptosis, metastasis, or escape from immune surveillance. Tumor associated
antigens are
generally proteins or polypeptides derived therefrom, but can be glycans,
lipids, or other small
organic molecules. Additionally, a tumor antigen can arise through increases
or decreases in
post-translational processing exhibited by a cancer cell compared to a normal
cell, for example,
protein glycosylation, protein lipidation, protein phosphorylation, or protein
acetylation. In
certain embodiments, the target antigen binding domain specifically binds to a
tumor associated
antigen selected from the group consisting of CD5, CD19, CD20, CD25, CD37,
CD30, CD33,
CD45, CAMPATH-1, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1,
folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA),
ferritin, GD2,
GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2
receptor,
fibroblast activation protein, tenascin, a metalloproteinase, endosialin,
vascular endothelial
growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2/neu, MAGE A3,
p53
nonmutant, NY-ESO-1, MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-
abl,
tyrosinase, survivin, PSA, hTERT, a Sarcoma translocation breakpoint protein,
EphA2, PAP,
ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic
acid, MYCN,
RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe(animal),
CYP1B1,
PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic
anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE
1, B7-H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2,
CMET, HER3, EPCAM, CA6, NAPI2B, TROP2, CLDN18.2, FAP, RON, LY6E, FRA, DLL3,
PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PD-L1, CD204, CD206,
CD301,
VTCN1, or VISTA.
[0334] In certain embodiments, the target antigen binding domain specifically
binds to a tumor
associated antigen selected from the group consisting of GD2, GD3, GM2, Ley,
polysialic acid,
fucosyl GM1, GM3, Tn, STn, sLe(animal), or GloboH or having an amino acid
sequence
comprising at least 80%, 90%, 95%, 97%, 98%, 99% or 100% homology to CD5,
CD19, CD20,
CD25, CD37, CD30, CD33, CD45, CAMPATH-1, HLD-DR, carcinoembryonic antigen
(CEA),
TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific
membrane
antigen (PSMA), ferritin, CA-125, CA19-9, epidermal growth factor, p185HER2,
IL-2 receptor,
tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor,
avB3, WT1, LMP2,
HPV E6, HPV E7, EGFRvIII, Her-2/neu, MAGE A3, p53 nonmutant, NY-ES0-1,
MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase,
survivin, PSA,
132
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
hTERT, a Sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP,
ERG, NA17,
PAX3, ALK, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, mesothelin (MSLN),
PSCA,
MAGE Al, CYP1B1, PLAV1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, Carbonic
anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE
1, B7-H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2,
CMET, HER3, EPCAM, CA6, NAPI2B, TROP2, CLDN18.2, fibroblast activation protein
(FAP),
RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR,
endoglin, PD-L1,
CD204, CD206, CD301, VTCN1, or VISTA. In certain embodiments, the target
antigen binding
domain specifically binds to a tumor associated antigen comprising Her2/Neu
(CD340). In
certain embodiments, the target antigen binding domain specifically binds to a
tumor associated
antigen comprising at least 80%, 90%, 95%, 97%, 98%, 99% or 100% homology to
CD5, CD19,
CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, HLD-DR, carcinoembryonic
antigen,
TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific
membrane
antigen, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9, epidermal growth
factor, p185HER2,
IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin,
metalloproteinases, endosialin,
vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-
2/neu,
idiotype, MAGE A3, p53 nonmutant, NY-ES0-1, PSMA, GD2, CEA, MelanA/MART1, Ras
mutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT,
Sarcoma
translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK,
androgen
receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1,
mesothelin, PSCA,
MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-
1,
RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17, LCK,
HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1,
PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EPCAM, MSLN, CA6, NAPI2B, TROP2,
CLDN18.2, FAP, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1,
VEGFR,
endoglin, PDL-1, CD204, CD206, CD301, VTCN1, or VISTA.
[0335] The antigen binding domain that specifically binds a tumor associated
antigen can be
derived from any known antibody with the ability to bind to an antigen shown
to be expressed by
cancer cells. In certain embodiments, the target antigen binding domain is
derived from an
antibody selected from the group of Etaracizumab (Abegrin), Tacatuzumab
tetraxetan,
Bevacizumab (Avastin), Labetuzumab, Cetuximab (Erbitux), Obinutuzumab
(Gazyva),
Trastuzumab (Herceptin), Clivatuzumab, Rituximab (MabThera, Rituxan),
Gemtuzumab of
Gemtuzumab ozogamicin (Mylotarg), Girentuximab (Rencarex), or Nimotuzumab
(Theracim,
Theraloc). In certain embodiments, the target antigen binding domain is
derived from
133
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Pertuzumab (Perjeta). A target antigen binding domain is derived from a known
antibody when it
comprises one or more CDR sequences identical to one or more CDR sequences of
the known
antibody. In certain embodiments, a target antigen binding domain is derived
from a known
antibody when it comprises three HCDR sequences identical to three HCDR
sequences of the
known antibody. In certain embodiments, a target antigen binding domain is
derived from a
known antibody when it comprises three LCDR sequences identical to three LCDR
sequences of
the known antibody.
[0336] In certain embodiments, the target antigen binding domain of the
recombinant bispecific
antibody is derived from pertuzumab. Pertuzumab is a monoclonal antibody that
specifically
binds the Her2/Neu antigen. The heavy chain amino acid sequence for pertuzumab
is set forth in
SEQ ID NO: 11; the light chain amino acid sequence for pertuzumab is set forth
in SEQ ID NO:
16. In certain embodiments, the target antigen binding domain comprises a CDR
sequence
identified from either of SEQ ID NO: 11 or SEQ ID NO: 16 identified using the
Kabat, IMGT or
Chothia method. In certain embodiments, the target antigen binding domain
comprises an amino
acid sequence with at least 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ
ID NO: 11. In
certain embodiments, the target antigen binding domain comprises an amino acid
sequence with
at least 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 16. In
certain embodiments,
the target antigen binding domain comprises an amino acid sequence with at
least 80%, 90%,
95%, 98%, 99%, or 100% identity to SEQ ID NO: 11 and SEQ ID NO: 16.
[0337] The heavy chain variable region amino acid sequence for pertuzumab is
set forth in SEQ
ID NO: 12; the light chain variable region amino acid sequence for pertuzumab
is set forth in
SEQ ID NO: 17. In certain embodiments, the target antigen binding domain
comprises a CDR
sequence identified from either of SEQ ID NO: 12 or SEQ ID NO: 17 identified
using the Kabat,
IMGT or Chothia method. In certain embodiments, the target antigen binding
domain comprises
an amino acid sequence with at least 80%, 90%, 95%, 98%, 99%, or 100% identity
to SEQ ID
NO: 12. In certain embodiments, the target antigen binding domain comprises an
amino acid
sequence with at least 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:
17. In
certain embodiments, the target antigen binding domain comprises an amino acid
sequence with
at least 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 12 and SEQ ID
NO: 17.
[0338] In certain embodiments, the target antigen binding domain may comprise
the CDR
sequence of pertuzumab. The amino acid sequence of the HCDR1 of pertuzumab is
set forth in
SEQ ID NO: 13; the amino acid sequence of the HCDR2 of pertuzumab is set forth
in SEQ ID
NO: 14; the amino acid sequence of the HCDR3 of pertuzumab is set forth in SEQ
ID NO: 15;
the amino acid sequence of the LCDR1 of pertuzumab is set forth in SEQ ID NO:
18; the amino
134
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
acid sequence of the LCDR2 of pertuzumab is set forth in SEQ ID NO: 19; and
the amino acid
sequence of the LCDR3 of pertuzumab is set forth in SEQ ID NO: 20. In certain
embodiments,
the target antigen binding domain comprises any one of SEQ ID NO: 13 ¨ SEQ ID
NO: 15 and
SEQ ID NO: 18 ¨ SEQ ID NO: 20. In certain embodiments, the target antigen
binding domain
comprises all of SEQ ID NO: 13¨ SEQ ID NO: 15 and SEQ ID NO: 18¨ SEQ ID NO:
20. In
certain embodiments, the target antigen binding domain comprises any one of
SEQ ID NO: 13 ¨
SEQ ID NO: 15. In certain embodiments, the target antigen binding domain
comprises any one
of SEQ ID NO: 18 ¨ SEQ ID NO: 20. In certain embodiments, the target antigen
binding domain
comprises a sequence at least all of SEQ ID NO: 13 and SEQ ID NO: 15. In
certain embodiments,
the target antigen binding domain comprises all of SEQ ID NO: 18 and SEQ ID NO
20. The
CDR sequences above may be incorporated into the target antigen binding domain
by any
method of recombinant DNA technology. The incorporated CDRs can have any
amount of amino
acid sequence identity to any one of SEQ ID NO: 13 ¨ SEQ ID NO: 15 and SEQ ID
NO: 18 ¨
SEQ ID NO: 20 that still retains the specific binding of pertuzumab, for
example, 80%, 90%,
95%, 98%, 99%, or 100% identity.
[0339] In certain embodiments, the recombinant bispecific antibody comprises a
target antigen
binding domain and an Fc region that are attached in a single polypeptide,
resulting in a fusion.
In certain embodiments, the recombinant bispecific antibody comprises a target
antigen binding
domain and an Fc comprising domain as a single polypeptide, which is a fusion.
In certain
embodiments, the target antigen binding domain attached to the Fc region
comprises an amino
acid sequence set forth in SEQ ID NO: 1311. In certain embodiments, the target
antigen binding
domain attached to the Fc region comprises an amino acid sequence at least
80%, 90%, 95%,
98%, 99%, or 100% identical to that set forth in SEQ ID NO: 1311.
[0340] In certain embodiments, the recombinant bispecific antibody comprises a
target antigen
binding with at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to 6,
7, 8, 9, 10, 11,
12 or more contiguous amino acids between amino acid 20 and amino acid 110 of
SEQ ID NO:
12 and at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to 6,7, 8,
9, 10, 11, 12 or
more contiguous amino acids between amino acid 20 and amino acid 105 of SEQ ID
NO: 17.
[0341] In some embodiments, the target antigen binding domain comprises an
immunoglobulin
heavy chain variable region or antigen binding fragment thereof and an
immunoglobulin light
chain variable region or antigen binding fragment thereof.
[0342] In some embodiments, the target antigen binding domain comprises a
single chain
variable region fragment (scFv).
135
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0343] In some embodiments, the target antigen binding domain comprises the
following CDRs:
a) HCDR1 comprising an amino acid sequence of SEQ ID NO: 13; b) HCDR2
comprising an
amino acid sequence of SEQ ID NO: 14; c) HCDR3 comprising an amino acid
sequence of SEQ
ID NO: 15; d) LCDR1 comprising an amino acid sequence of SEQ ID NO: 18; e)
LCDR2
comprising an amino acid sequence of SEQ ID NO: 19; and f) LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 20; and wherein the recombinant bispecific antibody
specifically binds
to Her2/neu or p185HER2.
[0344] In some embodiments, the target antigen binding domain comprises: a) a
VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
12; and b) a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 17. In
some embodiments, the target antigen binding domain comprises: a) a heavy
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
11; and b) a
light chain sequence having at least 80% sequence identity to an amino acid
sequence of SEQ ID
NO: 16.
[0345] In some embodiments, the target antigen binding domain comprises at
least 80%
sequence identity to the amino acid sequence between amino acid 20 and amino
acid 110 of SEQ
ID NO: 12 and at least 80% sequence identity to the amino acid sequence
between amino acid 20
and amino acid 105 of SEQ ID NO: 17; and wherein the recombinant bispecific
antibody
specifically binds to Her2/neu or p185HER2.
Effector Antigen Binding Domain
[0346] The recombinant bispecific antibodies comprise an effector antigen
binding domain. The
effector antigen binding domain specifically binds to a molecule present on an
antigen presenting
cell (APC), such as a dendritic cell. Antigen presenting cells regulate immune
response by
priming, and/or sustaining cell mediated immunity by T cells (both helper and
cytotoxic T cells).
As such, attracting antigen presenting cells to tumors and cancerous cells has
the potential to
boost cancer immunity and provide an adjuvant to monoclonal antibody therapy.
This can be
achieved by either providing activating signals to the APC (e.g., agonizing a
costimulatory
molecule) or blocking inhibitory signals (e.g., antagonizing a checkpoint
inhibitor). In certain
embodiments, the molecule present on the antigen presenting cell comprises a
costimulatory
molecule or other molecule that results in activation of the APC upon ligand
binding. In certain
embodiments, the costimulatory molecule is CD40, OX4OL, 4-1BBL, DEC-205, CD36,
CD204,
MARCO, DC-SIGN, CLEC9A, CLEC12A, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206,
CD4, CD32A, CD16A, HVEM, CD32B, PD-L1, or BDCA-2. In certain embodiments, the
molecule present on the antigen presenting cell comprises a costimulatory
molecule or other
136
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
molecule that results in activation of the APC upon ligand binding. In certain
embodiments, the
costimulatory molecule is CD40, OX4OL, 4-1BBL, DEC-205, CD36, CD204, MARCO, DC-
SIGN, CLEC9A, CLEC12A, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD4, CD32A,
CD16A, HVEM, CD32B, PD-L1, BDCA-2, TNFR2, or TREM2. The effector antigen
binding
domain can be a ligand, agonist, or agonistic antibody that results in
induction of one or more
activation markers of the APC, for example, cytokine release, chemokine
release, increased
expression of cell surface molecules that engage T cells, including MHC class
I, MHC class II or
costimulatory molecules. Cytokine release can be measured by a cytokine
release assay.
Chemokine release can be measured by an ELISA immunoassay. Expression of cell
surface
molecules can be measured by FACS. In certain embodiments, the effector
antigen binding
domain is an agonist of CD40, CD80, CD86, OX4OL, 4-1BBL, DEC-205, CD36, CD204,
MARCO, DC-SIGN, CLEC9A, CLEC12A, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206,
CD4, CD32A, CD16A, HVEM, CD32B, PD-L1, or BDCA-2. In certain embodiments, the
effector antigen binding domain is an agonist of CD40, CD80, CD86, OX4OL, 4-
1BBL, DEC-
205, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC12A, CLEC5A, Dectin 1, Dectin 2,
CLEC10A, CD206, CD4, CD32A, CD16A, HVEM, CD32B, PD-L1, BDCA-2, TNFR2, or
TREM2. In certain embodiments, the effector antigen binding domain is TNFR2 or
TREM. In
certain embodiments, the effector antigen binding domain is an agonist of
CD40. In certain
embodiments, the effector antigen binding domain is derived from an agonist
antibody of CD40,
and possesses one or more CDR sequences derived from a CD40 agonistic
antibody, such as, CP-
870,893, APX005M, 3C3, 3G5, Dacetuzmumab and its non-fucosylated form SEA-
CD40, or Chi
Lob 7/4. The effector antigen binding domain can be a ligand, antagonist, or
antagonistic
antibody that antagonizes a checkpoint inhibitor and results in induction of
one or more
activation markers of the APC, for example, cytokine release, chemokine
release, increased
expression of cell surface molecules that engage T cells, including MHC class
I, MHC class II or
costimulatory molecules. Cytokine release can be measured by a cytokine
release assay.
Chemokine release can be measured by an ELISA immunoassay. Expression of cell
surface
molecules can be measured by FACS. In certain embodiments, the effector
antigen binding
domain is an antagonist of PD-L1, PD-L2, galectin-9, Indoleamine 2,3-
dioxygenase, or CD276.
In certain embodiments, the effector antigen binding domain may not be a
lipocalin mutein. In
certain embodiments, the effector antigen binding domain can be an antibody
antigen binding
domain.
[0347] Dendritic cells are key antigen presenting cells that participate in
anti-cancer immunity.
In certain embodiments, the effector antigen binding domain specifically binds
to an antigen
137
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
present on a dendritic cell. In certain embodiments, the antigen present on a
dendritic cell
comprises CD11b, CD11c, MHC class II molecules, CD40, CD80, CD86, OX4OL, DEC-
205, 4-
1BBL, DEC-205, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC12A, CLEC5A, Dectin 1,
Dectin 2, CLEC10A, CD206, CD4, CD32A, CD16A, HVEM, CD32B, PD-L1, or BDCA-2. In
certain embodiments, the effector antigen binding domain specifically binds to
a protein with at
least 80%, 90%, 95%, 97%, 98%, 99%, or 100% homology to CD11b, CD11c, MHC
class II
molecules, CD40, CD80, CD86, OX4OL, DEC-205, 4-1BBL, DEC-205, CD36, CD204,
MARCO, DC-SIGN, CLEC9A, CLEC12A, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206,
CD4, CD32A, CD16A, HVEM, CD32B, PD-L1, or BDCA-2. In certain embodiments, the
effector antigen binding domain specifically binds to a protein with at least
80%, 90%, 95%, 97%,
98%, 99%, or 100% homology to CD11b, CD11c, MHC class II molecules, CD40,
CD80, CD86,
OX4OL, DEC-205, 4-1BBL, DEC-205, CD36, CD204, MARCO, DC-SIGN, CLEC9A,
CLEC12A, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD4, CD32A, CD16A, HVEM,
CD32B, PD-L1, BDCA-2, TNFR2, or TREM2. In certain embodiments, the effector
antigen
binding domain binds to the molecule present on the antigen presenting cell
comprising at least
80%, 90%, 95%, 97%, 98%, 99%, or 100% homology to CD40, OX4OL, DEC-205, 4-
1BBL,
CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64,
CD32A, CD16A, HVEM, CD32B, PD-L1, or BDCA-2. In certain embodiments, the
effector
antigen binding domain binds to the molecule present on the antigen presenting
cell comprising
at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% homology to CD40, OX4OL, DEC-
205, 4-
1BBL, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206,
CD64, CD32A, CD16A, HVEM, CD32B, PD-L1, TNFR2, TREM2, or BDCA-2.
[0348] In some embodiments, the effector antigen binding domain of the
recombinant bispecific
antibody can have a different binding affinity for a molecule present on an
antigen presenting cell
in recombinant form compared to the binding affinity for the molecule present
on the antigen
presenting cell when the molecule is expressed by the antigen presenting cell.
As used herein, the
recombinant form of a molecule present on an antigen presenting cell (i.e.,
the recombinant form)
can describe the molecule in a form that is independent from the antigen
presenting cell, and
therefore is not present on a cell or a live cell. In some aspects, a binding
affinity of the effector
antigen binding domain of the recombinant bispecific antibody to the molecule
present on the
antigen presenting cell is decreased compared to a binding affinity of the
effector antigen binding
domain of an antibody that lacks the target antigen binding domain, and this
can occur even
when the binding affinity of the effector antigen binding domain of the
recombinant bispecific
antibody to the molecule present on the antigen presenting cell in a
recombinant form is similar
138
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
to the binding affinity of the effector antigen binding domain of an antibody
that lacks the target
antigen binding domain. Furthermore, a Kd of the binding affinity of the
effector antigen binding
domain of the recombinant bispecific antibody to the molecule present on the
antigen presenting
cell can be increased by two times, five times, ten times, fifty times, or one-
hundred times
compared to the binding affinity of the effector antigen binding domain of an
antibody that lacks
the target antigen binding domain. In some aspects, the Kd for binding of the
effector antigen
binding domain to the molecule present on the antigen presenting cell is less
than 20 nM, less
than 100 nM, or less than 500 nM.
[0349] The effect of the target antigen binding domain and the effector
antigen binding domain
together can be to cluster antigen presenting cells around cancerous cells and
at tumor sites
resulting in activation of the antigen presenting effector functions of these
cells. In certain
embodiments, a recombinant bispecific antibody density of greater than 1000,
2000, 3000, 4000,
5000, 6000, 7000, 8000, 9000, 10,000 or more per cell, resulting from
recombinant bispecific
antibody binding to the tumor associated antigen, induces signaling in the
antigen presenting cell.
Signaling and clustering can suitably be measured in vitro. Signaling can be
suitably measured
using a cell line expressing the tumor associated antigen bound by the target
antigen binding
domain, and primary antigen presenting cells isolated from a human subject.
Signaling can be
assessed as cytokine release, chemokine release, or increased expression of
cell surface markers.
Cytokine release can be measured by a cytokine release assay. Chemokine
release can be
measured by an ELISA immunoassay. Expression of cell surface molecules can be
measured by
FACS.
[0350] In certain embodiments, the effector antigen binding domain of the
recombinant
bispecific antibody disclosed herein is derived from monoclonal antibody CP-
870,893. CP-
870,893 is a monoclonal antibody that specifically binds to and agonizes CD40.
The heavy chain
amino acid sequence for CP-870,893 is set forth in SEQ ID NO: 1; the light
chain amino acid
sequence for CP-870,893 is set forth in SEQ ID NO: 6. In certain embodiments,
the effector
antigen binding domain comprises a CDR sequence identified from either of SEQ
ID NO: 1 or
SEQ ID NO: 6 identified using the Kabat, IMGT, or Chothia method. In certain
embodiments,
the effector antigen binding domain comprises an amino acid sequence with at
least 80%, 90%,
95%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In certain embodiments, the
effector
antigen binding domain comprises an amino acid sequence with at least 80%,
90%, 95%, 98%,
99%, or 100% identity to SEQ ID NO: 6. In certain embodiments, the effector
antigen binding
domain comprises amino acid sequences with at least 80%, 90%, 95%, 98%, 99%,
or 100%
identity to SEQ ID NO: 1 and SEQ ID NO: 6.
139
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0351] The heavy chain variable region amino acid sequence for CP-870,893 is
set forth in SEQ
ID NO: 2; the light chain variable region amino acid sequence for CP-870,893
is set forth in SEQ
ID NO: 7. In certain embodiments, the effector antigen binding domain
comprises a CDR
sequence identified from either of SEQ ID NOs: 2 or 7 identified using the
Kabat, IMGT, or
Chothia method. In certain embodiments, the effector antigen binding domain
comprises an
amino acid sequence with at least 80%, 90%, 95%, 98%, 99%, or 100% identity to
SEQ ID NO:
2. In certain embodiments, the effector antigen binding domain comprises an
amino acid
sequence with at least 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:
7. In certain
embodiments, the effector antigen binding domain comprises an amino acid
sequence with at
least 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 2 and SEQ ID NO:
7.
[0352] In certain embodiments, the effector antigen binding domain comprises
the CDR
sequences of monoclonal antibody CP-870,893. The amino acid sequence of the
HCDR1 of CP-
870,893 is set forth in SEQ ID NO: 3; the amino acid sequence of the HCDR2 of
CP-870,893 is
set forth in SEQ ID NO: 4; the amino acid sequence of the HCDR3 of CP-870,893
is set forth in
SEQ ID NO: 5; the amino acid sequence of the LCDR1 of CP-870,893 is set forth
in SEQ ID NO:
8; the amino acid sequence of the LCDR2 of CP-870,893 is set forth in SEQ ID
NO: 9; and the
amino acid sequence of the LCDR3 of CP-870,893 is set forth in SEQ ID NO: 10.
In certain
embodiments, the effector antigen binding domain comprises any one of SEQ ID
NO: 3 ¨ SEQ
ID NO: 5 and SEQ ID NO: 8 ¨ SEQ ID NO: 10. In certain embodiments, the
effector antigen
binding domain comprises all of SEQ ID NO: 3 ¨ SEQ ID NO: 5 and SEQ ID NO: 8 ¨
SEQ ID
NO: 10. In certain embodiments, the effector antigen binding domain comprises
any one of SEQ
ID NO: 3 ¨ SEQ ID NO: 5. In certain embodiments, the effector antigen binding
domain
comprises any one of SEQ ID NO: 8 ¨ SEQ ID NO: 10. In certain embodiments, the
effector
antigen binding domain comprises all of SEQ IDO NO: 3 ¨ SEQ ID NO: 5. In
certain
embodiments, the effector antigen binding domain comprises all of SEQ ID NO: 8
¨ SEQ ID NO:
10. The CDR sequences above may be incorporated into the effector antigen
binding domain by
any method of recombinant DNA technology. The incorporated CDRs can have any
amount of
amino acid sequence identity to any one of SEQ ID NO: 3 ¨ SEQ ID NO: 5 and SEQ
ID NO: 8 ¨
SEQ ID NO: 10 that still retains the specific binding of CP-870,893, for
example, 80%, 90%,
95%, 98%, 99%, or 100% identity.
[0353] In certain embodiments, the effector antigen binding domain comprises a
CD40 scFv. The
CD40 scFv can be either attached (fused via a peptide bond between amino
acids) or coupled (via
a linker) to the C-terminus of the Fc comprising domain polypeptide. In
certain embodiments, the
CD40 scFv comprises an amino acid sequence set forth in SEQ ID NO: 1312. In
certain
140
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
embodiments, the effector antigen binding domain attached to the Fc comprising
domain
comprises an amino acid sequence at least 80%, 90%, 95%, 98%, 99%, or 100%
identical to that
set forth in SEQ ID NO: 1312.
[0354] In certain embodiments, the recombinant bispecific antibody comprises
an effector
antigen binding with at least 80%, 90%, 95%, 98%, 99%, or 100% sequence
identity to 6, 7, 8, 9,
10, 11, 12 or more contiguous amino acids between amino acid 20 and amino acid
110 of SEQ
ID NO: 12 and at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to
6, 7, 8, 9, 10,
11, 12 or more contiguous amino acids between amino acid 20 and amino acid 105
of SEQ ID
NO: 17.
[0355] In some embodiments, the effector antigen binding domain comprises an
immunoglobulin
heavy chain variable region or antigen binding fragment thereof and an
immunoglobulin light
chain variable region or antigen binding fragment thereof. In some
embodiments, the effector
antigen binding domain comprises a single chain variable region fragment
(scFv).
[0356] In some embodiments, the scFv comprises at least 80% sequence identity
to an amino
acid sequence of SEQ ID NO: 1312.
[0357] In some embodiments, the antigen presenting cell is a dendritic cell.
In some
embodiments, the antigen on the antigen presenting cell is a costimulatory
molecule.
[0358] In some embodiments, the antigen on the antigen presenting cell is
selected from the
group consisting of CD40, OX4OL, DEC-205, 4-1BBL, CD36, CD204, MARCO, DC-SIGN,
CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-
L1, or BDCA-2. In some embodiments, the effector antigen binding domain is a
CD40 agonist.
[0359] In some embodiments, the effector antigen binding domain comprises the
following
CDRs: a) HCDR1 comprising an amino acid sequence of SEQ ID NO: 3; b) HCDR2
comprising
an amino acid sequence of SEQ ID NO: 4; c) HCDR3 comprising an amino acid
sequence of
SEQ ID NO: 5; d) LCDR1 comprising an amino acid sequence of SEQ ID NO: 8; e)
LCDR2
comprising an amino acid sequence of SEQ ID NO: 9; and f) LCDR3 comprising an
amino acid
sequence of SEQ ID NO: 10.
[0360] In some embodiments, the effector antigen binding domain comprises: a)
a VH sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
2; and b) a VL
sequence having at least 80% sequence identity to an amino acid sequence of
SEQ ID NO: 7. In
some embodiments, the effector antigen binding domain comprises: a) a heavy
chain sequence
having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:
1; and b) a light
chain having at least 80% sequence identity to an amino acid sequence of SEQ
ID NO: 6.
[0361] In some embodiments, the antigen on the antigen presenting cell is
TREM2 or TNFR2.
141
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0362] In some embodiments, the effector antigen binding domain has an
increased binding
affinity to the antigen on the antigen presenting cell as compared to the
binding affinity of the
effector antigen binding domain of an antibody that lacks the target antigen
binding domain.
[0363] In some embodiments, a Kd of the binding affinity of the effector
antigen binding domain
of the recombinant bispecific antibody to the antigen on the antigen
presenting cell is increased
by two times, five times, ten times, fifty times, or one-hundred times
compared to the binding
affinity of the effector antigen binding domain of an antibody that lacks the
target antigen
binding domain.
[0364] In some embodiments, a Kd for binding of the effector antigen binding
domain to the
antigen on the antigen presenting cell is less than 20 nM, less than 100 nM,
or less than 500 nM.
Nucleotides Encoding Antibodies
[0365] In certain embodiments, antibodies are encoded by polynucleotides for
expression and
purification from a cell based system. Suitable polynucleotides include
vectors such as DNA
plasmids, viral vectors, and RNA molecules. Suitable viral vectors include
retroviral, lentiviral,
adenoviral, or baculoviral vectors.
[0366] Antibodies can be produced by a suitable method including by synthesis,
in a cell based
system, or a combination thereof. A suitable cell line for the production of
the recombinant
antibodies includes the CHO (Chinese hamster ovary) cell line or
variants/derivatives thereof.
Other suitable cell lines include AGELHN, NSO, 5p2/0, BHK21, HEK-293, HT-1080,
PER.C6,
HKB-11, CAP, and HuH-7 cell lines or variants/derivatives thereof. In a
certain embodiment, the
cell(s) utilized for production is transiently transfected or infected with
the vector(s). In a certain
embodiment, the production cell(s) is stably transfected with the vector(s),
and constitutes a
master cell bank for the production of antibodies. A master cell bank allows
freezing and
preservation of an antibody producing cell line. This allows for more
efficient and consistent
production of antibodies. Cells that are transfected or infected with
vector(s) encoding an
antibody are then cultured in a growth media for at least 1, 3, 5, 7, 9, 11,
14 days or more and the
growth media is harvested for purification of the antibody. In certain
embodiments, the growth
media lacks serum of human or animal origin.
[0367] In certain embodiments, antibodies isolated or purified after secretion
from a cell based
system. Purification includes at least one step comprising centrifugation,
precipitation, filtration,
dialysis. The Fc region of the antibodies may retain the ability to interact
with bacterial super
antigens such as Protein A or Protein G. In certain embodiments, purification
comprises a step
utilizing Protein A, Protein G, or a combination thereof, to specifically
separate recombinant
142
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
antibodies from other secreted proteins and serum. In certain embodiments,
column
chromatography is utilized.
Immune-Stimulatory Compounds
[0368] The antibody constructs and recombinant bispecific antibodies described
herein can
further be attached to an immune-stimulatory compound to form a conjugate. The
immune-
stimulatory compound can provide a direct, indirect or adjuvant effect. In
certain embodiments,
the immune-stimulatory compound can be coupled to the antibody construct, such
as to the Fc
domain of the antibody construct. An immune-stimulatory compound can be any
compound that
directly or indirectly stimulates an anti-tumor immune response after
administration. For example,
an immune-stimulatory compound can directly stimulate an anti-tumor immune
response by
causing the release of cytokines by its target cell, which results in the
activation of immune cells.
As another example, an immune-stimulatory compound can indirectly stimulate an
immune
response by suppressing IL-10 production and secretion by the target cell
and/or by supressing
the activity of regulatory T cells, resulting in an increased anti-tumor
response by immune cells.
The stimulation of an immune response by an immune-stimulatory compound can be
measured
by the upregulation of proinflammatory cytokines and/or increased activation
of immune cells.
This effect can be measured in vitro by co-culturing immune cells with cells
targeted by the
immune-stimulatory conjugate and measuring cytokine release, chemokine
release, proliferation
of immune cells, upregulation of immune cell activation markers, and/or ADCC.
ADCC can be
measured by an ADCC assay, which can determine the percentage of remaining
target cells, such
as tumor cells, in the co-culture after administration of the immune-
stimulatory conjugate with
the target cells and immune cells.
[0369] In certain embodiments, an immune-stimulatory compound can target a
pattern
recognition receptor (PRR). PRRs can recognize pathogen-associated molecular
patterns
(PAMPs) and damage-associated molecular patterns (DAMPs). A PRR can be
membrane bound.
A PRR can be cytosolic. A PRR can be a toll-like receptor (TLR). A PRR can be
RIG-I-like
receptor. A PRR can be a receptor kinase. A PRR can be a C-type lectin
receptor. A PRR can be
a NOD-like receptor. A PRR can be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,
TLR8,
TLR9, TLR10, TLR11, TLR12 or TLR13. A PRR can be TLR1, TLR2, TLR3, TLR4, TLR5,
TLR6, TLR7, TLR8, TLR9, and TLR10.
[0370] In certain embodiments, the immune-stimulatory compound can be a Damage-
Associated
Pattern Molecule (DAMP) or a Pathogen-Associated Molecular Pattern Molecule
(PAMP).
Immune-stimulatory molecular motifs, such as PAMPs, can be recognized by
receptors of the
innate immune system, such as Toll-like receptors (TLRs), Nod-like receptors,
C-type lectins,
143
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
and RIG-I-like receptors. These receptors can be transmembrane and intra-
endosomal proteins
which can prime activation of the immune system in response to infectious
agents such as
pathogens. Similar to other protein families, TLRs can have many isoforms,
including TLR4,
TLR7 and TLR8. TLR agonists can range from simple molecules to complex
macromolecules.
Likewise, the sizes of TLR agonists can range from small to large. TLR
agonists can be synthetic
or biosynthetic agonists. TLR agonists can also be PAMPs. Additional immune-
stimulatory
compounds, such as cytosolic DNA and unique bacterial nucleic acids called
cyclic dinucleotides,
can be recognized by Interferon Regulatory Factor (IRF) or stimulator of
interferon genes
(STING), which can act a cytosolic DNA sensor. Compounds recognized by
Interferon
Regulatory Factor (IRF) can play a role in immunoregulation by TLRs and other
pattern
recognition receptors.
[0371] Imiquimod, a synthetic TLR7 agonist, is currently approved for human
therapeutic
applications. Contained in a cream and marketed under the brand name AldaraTM,
imiquimod
serves as a topical treatment for a variety of indications with immune
components, such as,
actinic keratosis, genital warts, and basal cell carcinomas. In addition,
imiquimod is indicated as
a candidate adjuvant for enhancing adaptive immune responses when applied
topically at an
immunization site.
[0372] Another type of immune stimulatory molecular motif, Damage-Associated
Molecular
Mattern molecules (DAMPs), can initiate and maintain an immune response
occurring as part of
the non-infectious inflammatory response. DAMPs can be specially localized
proteins that, when
detected by the immune system in a location other than where DAMPs should be
located,
activate the immune system. Often, DAMPs can be nuclear or cytosolic proteins
and upon release
from the nucleus or cytosol, DAMP proteins can become denatured through
oxidation. Examples
of DAMP proteins can include chromatin-associated protein high-mobility group
box 1
(HMGB1), S100 molecules of the calcium modulated family of proteins and also
glycans, such
as hyaluronan fragments, and glycan conjugates. DAMPs can also be nucleic
acids, such as DNA,
when released from tumor cells following apoptosis or necrosis. Examples of
additional DAMP
nucleic acids can include RNA and purine metabolites, such as ATP, adenosine
and uric acid,
present outside of the nucleus or mitochondria.
[0373] In certain embodiments, an immune-stimulatory compound can be a Toll-
like receptor
agonist, a STING agonist, or a RIG-I agonist.
[0374] In certain embodiments, an immune-stimulatory compound can be a TLR
agonist.
Additionally, the immune response elicited by TLR agonists can further be
enhanced when co-
administered with a CD40-agonist antibody. For example, co-administration of a
TLR agonist
144
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
such as poly IC:LC with a CD40-agonist antibody can synergize to stimulate a
greater CD8+ T
cell response than either agonist alone.
[0375] In certain embodiments, the immune-stimulatory compound can be S-27609,
CL307, UC-
IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS-1463GS-9620,
GSK2245035,
TMX-101, TMX-201, TMX-202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-
052,
3M-854A, S-34240, KU34B, SB9200, SB11285, 8-substituted imidazo[1,5-
a]pyridine, or CL663.
[0376] In certain embodiments, the immune-stimulatory compound can be a TLR4
agonist, such
as AZ126 (N-(2-(cyclopentylamino)-2-oxo-1-(pyridin-4-yl)ethyl)-N-(4-
methoxypheny1)-3-
methyl-5-phenyl-1H-pyrrole-2-carboxamide) or AZ368 ((E)-3-(4-(2-
(cyclopentylamino)-1-(N-
(4-isopropylpheny1)-1,5-dipheny1-1H-pyrazole-3-carboxamido)-2-
oxoethyl)phenyl)acrylic acid).
[0377] In some embodiments, the immune-stimulatory compound can be a TLR7
agonist, such
as TLR7 agonist R848.
[0378] The immune-stimulatory compound can comprise an inhibitor of TGFB, Beta-
Catenin,
PI3K-beta, STAT3, IL-10, IDO or TDO. The immune-stimulatory compound can be an
inhibitor
of the beta-catenin pathway, such as an inhibitor of TNIK or Tankyrase. In
certain embodiments,
the immune-stimulatory compound be a kinase inhibitor. In certain embodiments,
the kinase
inhibitor can be an inhibitor of CDK4/6, such as, for example, abemaciclib or
palbociclib.
[0379] The immune-stimulatory compound can be LY2109761, G5K263771, iCRT3,
iCRT5,
iCRT14, LY2090314, CGX-1321, PRI-724, BC21, ZINCO2092166, LGK974, IWP2,
LY3022859, LY364947, SB431542, AZD8186, SD-208, indoximod (NLG8189), F001287,
GDC-0919, epacadostat (INCB024360), RG70099, 1-methyl-L-tryptophan,
methylthiohydantoin
tryptophan, brassinin, annulin B, exiguamine A, PIM, LM10, 8-substituted 2-
amino-3H-
benzo [b]azepine-4-carboxamide, or INCB023843.
[0380] In certain embodiments, the immune-stimulatory compound can be coupled
to the
antibody construct via a linker. In certain embodiments, the immune-
stimulatory compound is
coupled to the antibody construct using a linker wherein the immune-
stimulatory compound
comprises a Toll-like receptor agonist, STING agonist, or RIG-I agonist. In
certain embodiments,
the Toll-like receptor agonist comprises a CpG oligonucleotide, Poly G10, Poly
G3, Poly I:C,
Lipopolysaccharide, zymosan, flagellin, Pam3CSK4, PamCysPamSK4, dsRNA, a
diacylated
lipopeptide, a triacylated lipoprotein, lipoteichoic acid, or a peptidoglycan.
In certain
embodiments, the STING agonist comprises a cyclic dinucleotide. In certain
embodiments, the
RIG-1 agonist comprises a 5'ppp-dsRNA.
[0381] A PRR agonist can be pathogen-associated molecular pattern (PAMP)
molecule. A
PAMP molecule can be a toll-like receptor agonist. A PRR agonist can be a toll-
like receptor
145
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
agonist. A toll-like receptor agonist can be any molecule that acts as an
agonist to at least one
toll-like receptor. A toll-like receptor agonist can be bacterial lipoprotein.
A toll-like receptor
agonist can be bacterial peptidoglycans. A toll-like receptor agonist can be
double stranded RNA.
A toll-like receptor agonist can be lipopolysaccharides. A toll-like receptor
agonist can be
bacterial flagella. A toll-like receptor agonist can be single stranded RNA. A
toll-like receptor
can be CpG DNA. A toll-like receptor agonist can be imiquimod. A toll-like
receptor agonist can
be CL307. A toll-like receptor agonist can be S-27609. A toll-like receptor
agonist can be
resiquimod. A toll-like receptor agonist can be UC-IV150. A toll-like receptor
agonist can be
gardiquimod. A toll-like receptor agonist can be motolimod. A toll-like
receptor agonist can be
VTX-1463. A toll-like receptor agonist can be GS-9620. A toll-like receptor
agonist can be
GSK2245035. A toll-like receptor agonist can be TMX-101. A toll-like receptor
agonist can be
TMX-201. A toll-like receptor agonist can be TMX-202. A toll-like receptor
agonist can be
isatoribine. A toll-like receptor agonist can be AZD8848. A toll-like receptor
agonist can be
MEDI9197. A toll-like receptor agonist can be 3M-051. A toll-like receptor
agonist can be 3M-
852. A toll-like receptor agonist can be 3M-052. A toll-like receptor agonist
can be 3M-854A. A
toll-like receptor agonist can be S-34240. A toll-like receptor agonist can be
CL663. A RIG-I
agonist can be KIN1148. A RIG-I agonist can be SB-9200. A RIG-I agonist can be
KIN700,
KIN600, KIN500, KIN100, KIN101, KIN400, or KIN2000. A toll-like receptor
agonist can be
KU34B.
[0382] A PRR agonist can be a damage-associated molecular pattern (DAMP)
molecule. A
DAMP molecule can be an intracellular protein. A DAMP molecule can be a heat-
shock protein.
A DAMP molecule can be an HMGB1 protein. A DAMP molecule can be a protein
derived from
the extracellular matrix that is generated after tissue injury. A DAMP
molecule can be a
hyaluronan fragment. A DAMP molecule can be DNA. A DAMP molecule can be RNA. A
DAMP molecule can be an S100 molecule. A DAMP molecule can be nucleotides. A
DAMP
molecule can be an ATP. A DAMP molecule can be nucleosides. A DAMP molecule
can be an
adenosine. A DAMP molecule can be uric acid.
[0383] Additionally, an immune-stimulatory compound can target stimulator of
interferon genes
(STING). STING can act as a cytosolic DNA sensor wherein cytosolic DNA and
unique bacterial
nucleic acids called cyclic dinucleotides are recognized by STING, and
therefore STING
agonists. Interferon Regulatory Factor (IRF) agonist can be KIN-100. Non-
limiting examples of
STING agonists include:
146
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
, 0
_x1 p49
/ N% 0
X3 0
0 X4
0
OH: +
X6
0....,...p........ 0 OX b
0 \ _
0 X2
, wherein in some embodiments,
Xi=X2=0; X3=G; X4=G; X5=CO(CH2)12CH3; X6=2 TEAH; in some embodiments, Xi=X2=S
[Rp,Rp]; X3=G; X4=A; X5=H; X6=2 TEAH; in some embodiments, Xi=X2=S [Rp,Rp] ;
X3=A;
X4=A; X5=H; X6=2 Na; in some embodiments, Xi=X2=S [Rp,Rp] ; X3=A; X4=A; X5=H;
X6=2
NH4; and in some embodiments, Xi=X2=0 ; X3=G; X4=A; X5=H; X6=2 TEAH,
P: ¨ . ; tio2
i
41 43,-sss<
a=(. .,,,;% N :13.,==-õ4\ 1 04f.. 14
.õ0 P
-.-..,,-4 ,:õ,:õ.õ õ 3.3*i ".7"t'-f¨ . , le i =q
. ti=sulfro h , 31.t
s. \ . t oe. '''s c; I Ak. 0 ,eRs". 0 e t 1 3 i
, ;zie s=",t3 =\' 1 Z
z,5 ...-=== . k ,.\
\'''''' +%."1 f.3 \ I "..-Sitf<::: '''''<...". 441===========c
r:s. i= = *=.'1µ NN:z \'''.'14,*" . C` `'''' 111'.."'4=ti =
,1
...M2
---,...,,.,...... 6.,,, -,,,,,,..,0 ,5õ -0 s :.-
=&k,
# NA.
0 0 a 1 0 .s*
ai.42 :?w,k
4'..õ.1..
= A ::: g=¨=-4
? ¨i
1(,),
tt N
N Hz
<sc.
,.. ..,
1-KL-4.,,õ0 3.33t,. 44..a. *
===, =tp
....t.,,,,,, = 4
ti ..S...
, wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H;
Ri=H, R2=ally1;
Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl,
R2=H; Ri=H,
R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H,
R2=myristoyl;
Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
147
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
t, ... ,
',/
N 4 =
i.... ___________________________________________________ k -.1,....õ4=
...:
¨..,
:õ,: .:., .,,, , <i". 1 14 N \ :-.3 0=`'s -*
< 1 .,. 1 N \\ i==:.: rs''' ,$***=:*.t 4:'( 'I'' i .,,; \ ,:
k, . = \ ,...." \ ,,....1
.P, #
\
if---1
:, .1...s.f
6?1 \ 0- 4 ===,- // µ,...
-4,-
i't ?:t ti =,, is's,
ti,ai---K:µ p
tiKz
1õ,
/
\ === .1
, ( - %qr..
!..,,.. ;X)
' .4'
0, ,....)....µoz.k. i
7 : , µ=.$ %.18.z
, 1
,
wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H,
R2=ally1;
Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl,
R2=H; Ri=H,
R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H,
R2=myristoyl;
Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
; N ii,:
N
/ 14.........< g======<,,
. ,'"'"µµ
iss, ?'''81
I -
---1 = / (5 \,..,...cl .,0
,., 1 0,...,3,/ t 1 , ,,,,i
e . ***-** . 'a ,,,- i ar¨ ,
......:.....,,t):::(\s_j:
N ; \ 'NO a b 0; 22'^0 ((' i .. 1
I,'
.4\Cf
'..,.... 0Rt
4 % 4 .
0 W 0 0- 0 =0
:CI, N ti2
,N,.....i
N iet. 1
N
')',"..."4 ' Z=:
e.
-4'
,õ ...........õ,,,,
13 ,
(kci
N '7
0
; ,
oR
0,,..,a...õ:3 ,
I,''
0 0. ti
,
wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H,
R2=ally1;
Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl,
R2=H; Ri=H,
148
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H,
R2=myristoyl;
Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
X
RI
¨ 0 0
R4 0
FRI 3
R2
, wherein each X is independently 0 or S, and R3
and R4 are each independently H or an optionally substituted straight chain
alkyl of from 1 to 18
carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from
1-9 carbons, an
optionally substituted alkynyl of from 1-9 carbons, or an optionally
substituted aryl, wherein
substitution(s), when present, may be independently selected from the group
consisting of C1,6
alkyl straight or branched chain, benzyl, halogen, trihalomethyl, Ci_6 alkoxy,
¨NO2, ¨NH2, ¨
OH, ¨0, ¨COOR - where R - is H or lower alkyl, ¨CH2OH, and ¨CONH2, wherein R3
and R4 are not both H,
9
B,
= i 4
õµõ
HO I
9
H
B.!
, wherein Xi=X2=0; Xi=X2=S; or X1=0 and X2=S,
149
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
NH3
N.., ..,...,".,,,
ci
i ,I i ,o, \
-0..........õ-- ----At t,------- ,-
i \\ ....-D, i --N
1 , ..,,,
i,
h1 \
i
t
-0
N.s://)
"\:', ...., ',..
T
N1-1.3
,
0
I 1
t\?,
11
i, ......,
-0 ............... P"-
1 \ .0õ 7 ----- 14 N1
,
õ....-- ..,..I
\ t i
\
QH
,.............N,N
i ----
tit3µ\. ----"."- '7
-- .N
! 1
I i
0 ,
NH:.
N,
' ""v"...\,` -'''= ':-.:14
11 _,A),õ
.., s ,,,,,,.....,
\ RP i ,o /
.
v..............,..:( N
HO /
O µ t
ICI \
0 N
PpI
..--<;.- N.
r.,-- N........¨ N. ----o-- z:-..z.:-..0
i \
N N..
s'=-.N, .õ...-"-`..., //
tkt ti 2.
,
150
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
0
N L
/....
R
ii , \0¨ i
"s--4i--- --\ N-------',,,,i,7-s-,
1 RP % ,...Ø..õ / "NH
Q
:-1c1 i
i µ
OH
ii........\.
Fil)/0
,....,,...,`" .......--N ' ..=-=-= k)
\
HN,N. õõ.....,...,,, /::::=. ''
!
I
,
N-H2
I
,N,,, ..,....-"Z=\,. \\,
1
I ......= 0 ,..,, \
\ i
i
.... \ ,...-,..
I/ --...,N,..
HO
1
ismilimmi \I I
,( /
i \
s,
0 N
N i "o"\ Sp
---"1. '...,.
--;.---- ^.... ,...._.¨ N """ 0 ---,P"......,, ... -
.,\ 6
N'N'\õ.
N..õ: ..."....w.
N H ?
,
0
/2 ....õ,...õ.
'N H
0
\ 1
H ,o--- i
... ..............
$ p---- ----\ \ 0 NH,
s ..,
," `.... i
0
HO
i
i
/ \
i 1 \
i
0 OH
/ .`",.. cy--'-' . i
Sp /
N.,.------ l';\
0- ------, , --...,s--
i
0
H N \
**\....,,,=-= "'''..... /
N
I
i
0
,
151
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0
N,
'1,,ii-i
I I \ õo, \
I
----... ----
0=p-:- --- -\
P \ v 0,--ij
6
1-i 0 i
µ i
2.Hµ \
0. N
= ;I
--1
HaN .õN. i ^'N.. 0' \ Stil
=--;' N,..,.._ N ,
1
8 ..,
0
N.
1
,and
N #12
Nõ
..õ...........,,,,,,,,t4
S"
\
*N HO ------'''N. .-',..-<----'
i
1µ..- Q1
1
0
i
Ississississis 1:\ I \
1 µ
i
0 0 H
N =-='"
...-N, i N'O'''' \_ Sp /
-- "'---N, _ -----0---?1 s-
0
N -N,
i
1
N
[0384] An immune-stimulatory compound can be a PRR agonist. An immune-
stimulatory
compound can be a PAMP. An immune-stimulatory compound can be a DAMP. An
immune-
stimulatory compound can be a TLR agonist. An immune-stimulatory compound can
be a
STING agonist. An immune-stimulatory compound can be a cyclic dinucleotide.
[0385] The specificity of the antigen-binding domain to an antigen of a
conjugate disclosed
herein can be influenced by the presence of an immune-stimulatory compound.
The antigen-
binding domain of the conjugate can bind to an antigen with at least about
10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%,
about 90%,
about 95%, or about 100% of a specificity of the antigen-binding domain to the
antigen in the
absence of the immune-stimulatory compound.
152
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0386] The specificity of the Fc domain to an Fc receptor of a conjugate
disclosed herein can be
influenced by the presence of an immune-stimulatory compound. The Fc domain of
the conjugate
can bind to an Fc receptor with at least about 10%, about 20%, about 30%,
about 40%, about
50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or
about 100% of a
specificity of the Fc domain to the Fc receptor in the absence of the immune-
stimulatory
compound.
[0387] The affinity of the antigen-binding domain to an antigen of a conjugate
disclosed herein
can be influenced by the presence of an immune-stimulatory compound. The
antigen-binding
domain of the conjugate can bind to an antigen with at least about 1%, about
5%, about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 85%,
about 90%, about 95%, or about 100% of an affinity of the antigen-binding
domain to the antigen
in the absence of the immune-stimulatory compound.
[0388] The affinity of the Fc domain to an Fc receptor of a conjugate
disclosed herein can be
influenced by the presence of an immune-stimulatory compound. The Fc domain of
the conjugate
can bind to an Fc receptor with at least about 1%, about 5%, about 10%, about
20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%,
about 95%,
or about 100% of an affinity of the Fc domain to the Fc receptor in the
absence of the immune-
stimulatory compound.
[0389] The Kd for binding of an antigen-binding domain to an antigen in the
presence of an
immune-stimulatory compound can be about 2 times, about 3 times, about 4
times, about 5 times,
about 6 times, about 7 times, about 8 times, about 9 times, about 10 times,
about 15 times, about
20 times, about 25 times, about 30 times, about 35 times, about 40 times,
about 45 times, about
50 times, about 60 times, about 70 times, about 80 times, about 90 times,
about 100 times, about
110 times, or about 120 times greater than the Kd for binding of the antigen
binding domain to
the antigen in the absence of the immune-stimulatory compound.
[0390] The Kd for binding of an Fc domain to a Fc receptor in the presence of
an immune-
stimulatory compound can be about 2 times, about 3 times, about 4 times, about
5 times, about 6
times, about 7 times, about 8 times, about 9 times, about 10 times, about 15
times, about 20 times,
about 25 times, about 30 times, about 35 times, about 40 times, about 45
times, about 50 times,
about 60 times, about 70 times, about 80 times, about 90 times, about 100
times, about 110 times,
or about 120 times greater than the Kd for binding of the Fc domain to the Fc
receptor in the
absence of the immune-stimulator compound.
[0391] Affinity can be the strength of the sum total of noncovalent
interactions between a single
binding site of a molecule, for example, an antibody, and the binding partner
of the molecule, for
153
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
example, an antigen. The affinity can also measure the strength of an
interaction between an Fc
portion of an antibody or antibody construct and the Fc receptor. Unless
indicated otherwise, as
used herein, "binding affinity" can refer to intrinsic binding affinity which
reflects a 1:1
interaction between members of a binding pair (e.g., antibody and antigen or
Fc domain and Fc
receptor). The affinity of a molecule X for its partner Y can generally be
represented by the
dissociation constant (Kd). Affinity can be measured by common methods known
in the art,
including those described herein. Specific illustrative and exemplary
embodiments for measuring
binding affinity are described in the following.
[0392] In some embodiments, an antibody or antibody construct provided herein
can have a
dissociation constant (Kd) of about 1 t.M, about 100 nM, about 10 nM, about 5
nM, about 2 nM,
about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, or about
0.001 nM or
less (e.g., 10-8 M or less, e.g., from 10-8M to 10-13 M, e.g., from 10-9 M to
10-13 M). An affinity
matured antibody can be an antibody with one or more alterations in one or
more
complementarity determining regions (CDRs), compared to a parent antibody,
which may not
possess such alterations, such alterations resulting in an improvement in the
affinity of the
antibody for antigen. These antibodies can bind to their antigen with a Kd of
about 5x10-9 M,
about 2x10-9 M, about 1x10-9 M, about 5x10-1 M, about 2x10-9 M, about 1x10-1
M, about 5x10-
ii
M, about 1x10- 1 1 M, about 5x10-12 M, about 1x10-12 M, or less. In some
embodiments, the
conjugate can have an increased affinity of at least 1.5-fold, 2-fold, 2.5-
fold, 3-fold, 4-fold, 5-
fold, 10-fold, 20-fold, or greater as compared to a conjugate without
alterations in one or more
complementarity determining regions.
[0393] Kd can be measured by any suitable assay. For example, Kd can be
measured by a
radiolabeled antigen binding assay (RIA). For example, Kd can be measured
using surface
plasmon resonance assays (e.g., using a BIACOREC)-2000 or a BIACOREC)-3000).
[0394] Agonism can be described as the binding of a chemical to a receptor to
induce a
biological response. A chemical can be, for example, a small molecule, a
compound, or a protein.
An agonist causes a response, an antagonist can block the action of an
agonist, and an inverse
agonist can cause a response that is opposite to that of the agonist. A
receptor can be activated by
either endogenous or exogenous agonists.
[0395] The molar ratio of a conjugate refers to the average number of immune-
stimulatory
compounds conjugated to the antibody construct in a preparation of a
conjugate. The molar ratio
can be determined, for example, by Liquid Chromatography/Mass Spectrometry
(LC/MS), in
which the number of immune-stimulatory compounds conjugated to the antibody
construct can
be directly determined. Additionally, as non-limiting examples, the molar
ratio can be
154
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
determined based on hydrophobic interaction chromatography (HIC) peak area, by
liquid
chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-
MS), by UV/Vis
spectroscopy, by reversed-phase-HPLC (RP-HPLC), or by matrix-assisted laser
desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).
[0396] In some embodiments, the molar ratio of immune-stimulatory compound to
antibody
construct can be less than 8. In other embodiments, the molar ratio of immune-
stimulatory
compound to antibody construct can be 8, 7, 6, 5, 4, 3, 2, or 1.
[0397] In some aspects, the present disclosure provides an immune-stimulatory
compound
represented by the structure of Formula (I):
0 X1
/ ,
p-u
/ B1
o
0
B2 /0/\N1(R1)2
0-P
/ 0
X2
(I)
or a pharmaceutically acceptable salt thereof, wherein:
Xl is selected from -0R2 and -SR2;
X2 is selected from -0R3 and -SR3;
B1 and B2 are independently selected from optionally substituted nitrogenous
bases;
Y is selected from -0124, -NR4R4, and halogen;
121, R2, R3 and R4 are independently selected at each occurrence from
hydrogen, -
c(=o)Rioo, _
C(=0)0Rim and -C(=0)NRim; Ci_10 alkyl, C2-10 alkenyl, C2-10 alkynyl, each of
which is independently optionally substituted at each occurrence with one or
more substituents
selected from halogen, -ORm , -swoo, _N(Rioo)2, _s(0)Rioo, -S(0)2R' ,
_c(0)Rioo, _
C(0)0R1 ,
-0C(0)R1 , -NO2, =0, =s, =N(Rioo), _
P(0)(0R1 )2, -0P(0)(0R1 )2, -CN, C3_10 carbocycle and
3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered
heterocycle,
wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in 121, R2, R3
and R4 is
independently optionally substituted with one or more substituents selected
from halogen, -
()R' , -SR100, _N(R100)2, _s(o)R100, _s(0)2R100 _c(o)R100, _
C(0)0R1 , -0C(0)R1 , -NO2, =0,
=s, =N(R100), _
P(0)(0R1 )2, -0P(0)(0Rim)2, -CN, C1_6 alkyl, C2_6alkenyl, and C2_6alkynyl;
and
Rm at each occurrence is independently selected from hydrogen; and C1_10
alkyl, C2-10
alkenyl, C2_10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle
each of which is
155
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, =S, and haloalkyl.
[0398] In some embodiments, the compound of Formula (I) is represented by
Formula (IA):
X1
0 1
¨0
ZP
\
y 0 B1
0
B2 Ck c---7N/
0 N(R1)2
0¨p
/ 0
x2
(IA) ,
or pharmaceutically acceptable salts thereof.
[0399] In an alternative embodiment, the compound of Formula (I) is
represented by Formula
(TB):
X1
0 I
P-0
7
B1
0
B-Ck NH(R1)2 0
0------- 7-...
/F)
X2 0
(IB) ,
or a pharmaceutically acceptable salt thereof.
[0400] In various embodiments, B1 and B2 are independently selected from
optionally substituted
N'N
I ,
Lz.,.. ,......---
N N
purines. In certain embodiments, B1 and B2 are independently selected from:
H . in
certain embodiments, B1 and B2 are independently selected from optionally
substituted
pyrimidines.
[0401] In some embodiments, optionally substituted purines may include
optionally substituted
adenine, optionally substituted guanine, optionally substituted xanthine,
optionally substituted
hypoxanthine, optionally substituted theobromine, optionally substituted
caffeine, optionally
substituted uric acid, and optionally substituted isoguanine. In certain
embodiments, B1 and B2
are independently selected from:
156
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
0 CH3 0 CH3
NH2 0 0 0 H
HNJLNA H3C.N)1NNi
\
N.....,)N N--...A H1\11).EN N"---ANI-1 I // I
K" I) X-1 0 N--N 0 N -
'sN
NN N---N NH2 ON N N---N I I
H H H H , CH3 CH3
, ,
0 NH2
H
ii
N3NH
N--- NH
I
NNO \N- N'L0
H H ,and H ,optionally substituted by one or more additional
substituents.
NH2
N--...)N
I
N"--.N
[0402] In certain embodiments, B1 and B2 are independently selected from: -4,,
,
0 0 0 0 NH2
NDNH I N N--...)L LA 1-1y). ) y1-I 0 1 r
N----NH
\ _...,
N, N NH2 ON N N"--.N N--ec, N" -Ncl
H .,,L, ,,,L , 1- H , and 4A, , wherein the
point of connectivity of Blto the remainder of the compound is represented by
1 .
[0403] In a preferred embodiment, B1 and B2 are independently selected from
optionally
substituted adenine and optionally substituted guanine. In certain
embodiments, B1 and B2 are
NH2 0
N.....,) N --Amu
N
I 1 3..:
"--Nr
independently selected from: H , and --
N N - N NH
I-I 2,
optionally further substituted by
one or more substituents. In certain embodiments, B1 and B2 are independently
selected from:
NH2 0
N--...)N N.--ANH
I ) I
N-Th\r
/N---N NH2
/
-1-t, and "-q-L- .
[0404] In some embodiments, B1 and B2 are independently optionally substituted
with one or
more substituents, wherein the optional substituents on B1 and B2 are
independently selected at
each occurrence from halogen, =0, =S, -Oleo, _swoo, _N(Rioo)2, _s(0)Rioo,
_s(0)2Rioo, _
c(0)Rioo, _
C(0)0Rioo, -0C(0)Rioo, -NO2,
.2 _
-P(0)(0R1oo), OP(0)(0Rin2 and -CN; C1_10 alkyl,
C2_10 alkenyl, C2_10 alkynyl, each of which is independently optionally
substituted at each
occurrence with one or more substituents selected from halogen, -ORloo, _swoo,
_N(Rioo)2, _
S(0)R' , -S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0Rim)2, -OP (0)(0R1m)2, -CN, C3-10 carbocycle and 3- to 10-membered
157
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
heterocycle; and C3_10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3_10
carbocycle and 3- to 10-membered heterocycle is independently optionally
substituted with one
or more substituents selected from halogen, -0Rioo, _sec), _N(Rioo)2,
_s(0)Rioo, _s(0)2Rioo _
C(0)R' , _
C(0)0Rioo, -0C(0)R1 , -NO2, =0, =S,
=N(Rioo),
-P(0)(ORMI:2, -OP )
(0)(0R1m)2, -CN, C1_6 alkyl, C2lk _6 aenyl, and C2_6 alkynyl.
k
[0405] In certain embodiments, B1 and B2 are independently optionally
substituted with one or
more substituents, wherein the optional substituents on B1 and B2 are
independently selected at
each occurrence from halogen, =0, =S, -ORM, -SR100, _N(R100)2, _s(o)R100,
_s(o)2R100,
C(0)R' ,
C(0)0R100, -0C(0)R'
,
NO2, oos2 _
-P(0)(0R1 ), OP(0)(0R1 )2, -CN and C1_10 alkyl.
[0406] In some embodiments, B1 is an optionally substituted guanine. In
certain embodiments,
0 0
NH
Ii
I
I\r-N NH2 NN 2
B1 is H . In certain embodiments, B1 is ¨I- ,
wherein the point of
connectivity of B1 to the remainder of the compound is represented by . In
some
NH2
NN
I ,j
NN
embodiments, B1 is an optionally substituted adenine. In certain embodiments,
B1 is H
NH2
NN
In certain embodiments, B1 is ,
wherein the point of connectivity of B1 to the
remainder of the compound is represented by .
[0407] In some embodiments, B2 is an optionally substituted guanine. In
certain, embodiments,
0 0
NH NH
I I
2 1\r-N NH 2 NN 2
B is H . In certain embodiments, B2 is ¨I- ,
wherein the point of
connectivity on B2 is represented by . In some embodiments, B2 is an
optionally substituted
NH2 NH2
N NN
I I
NN
adenine. In certain embodiments, B2 is H . In certain embodiments, B2 is
dul,u
wherein the point of connectivity on B2 is represented by .
158
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0408] In some embodiments, B1 is an optionally substituted guanine and B2 is
an optionally
substituted guanine. In some embodiments, B1 is an optionally substituted
adenine and B2 is an
optionally substituted guanine.
[0409] In various embodiments, Xl is selected from ¨OH and ¨SH. For example,
Xl may be ¨
OH. In various embodiments, X2 is selected from ¨OH and ¨SH. For example, X2
may be ¨OH.
In some embodiments, Xl is ¨OH and X2 is ¨OH. In some embodiments, Xl is ¨SH
and X2 is ¨
SH.
[0410] In various embodiments, Y is selected from ¨OH, ¨0-Ci_10 alkyl,
¨NH(Ci_10 alkyl), and ¨
NH2. For example, Y may be ¨OH.
[0411] In various embodiments, le is independently selected at each
occurrence from hydrogen
and C1_10 alkyl optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, and =S.
[0412] In various embodiments, the compound of Formula (I) is represented by
Formula (IC):
OH 0
0 / N AF i
- P---
V 0,
H 0
0
0!, ` N N NH2
,01
H 2N *1\1,_N c:1 NH2
HIN 0 -
/ 0
HO (IC)
0 ,
or a pharmaceutically acceptable salt thereof. In some embodiments, the
compound of Formula
(IC) is represented by Formula (ID):
0.9H
) N61-1
HQ C) PO 3L N NH2
c
.: 7.
0 \i
/
0\ "
H2N *1\1 N /0 NH2
I-I N,_
ILN 0 ¨p
/ HO0 (ID)
0 ,
or a pharmaceutically acceptable salt thereof.
[0413] In various embodiments, the compound is a pharmaceutically acceptable
salt. In some
embodiments, the compound or salt is a modulator of a stimulator of interferon
genes (STING).
The compound or salt may agonize a stimulator of interferon genes (STING). In
certain
embodiments, the compound or salt may cause STING to coordinate multiple
immune responses
to infection, including the induction of interferons and STAT6-dependent
response and selective
159
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
autophagy response. In certain embodiments, the compound or salt may cause
STING to mediate
type I interferon production.
[0414] In some aspects, the present disclosure provides a compound represented
by the structure
of Formula (II):
0 X1
p-u
/ B1
Yo
oi, 0
N
B2 /
0-F)
/ 0
X2
(II)
or a pharmaceutically acceptable salt thereof, wherein:
Xl is selected from -0R2 and -SW;
X2 is selected from -0R3 and -Re;
B1 and B2 are independently selected from optionally substituted nitrogenous
bases,
wherein each optional substituent is independently selected from halogen, -
OW00, -SR100, _
N(Rioo)2; _s(0)Rioo; _s(0)2Rioo; _c(0)Rioo; _
C(0)0Rioo; -0C(0)Rim, -NO2, =0, =S,
=N(Rioo); _cN; R6, and _)(3;
Y is selected from -0124, -Re, -NR4R4, and halogen;
Z is selected from -0R5' -Re, and -NR5R5;
R', R2, R3, -4,
K and R5 are independently selected from a -X3; hydrogen, -C(=o)Rioo; _
C(=0)0Rim and -C(=0)NRin C1_10 alkyl, C2-10 alkenyl, C2-10 alkynyl, each of
which is
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -0R100, -SR100, _N(R100)2, _s(0)R100, _s(0)2R100, _c(0)R100, _
C(0)0R1M, -
OC(0)R100, -NO2, =0, =s, =N(R100),
-P(0)(0R1w)2, -0 P(0)(ORM)2, -CN, C3-10 carbocycle and
3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered
heterocycle,
wherein each C3_10 carbocycle and 3- to 10-membered heterocycle in 121, R2,
R3, R4, and R5 is
optionally substituted with one or more substituents selected from halogen, -
OW00, -SR100, _
N(Rioo)2; _s(0)Rioo; _s(0)2Rioo _c(0)Rioo; _
C(0)0Rioo; -0C(0)Rim, -NO2, =0, =S,
=N(Rioo);
-P(0)(0R1w)2, -0 P(0)(0R1m)2, -CN, C1_6 alkyl, C2-6 alkenyl, C2_6 alkynyl;
R6 is independently selected from -C(=o)Rioo; _
C(=0)0Rim and -C(=0)NRin Cl-lo
alkyl, C2-10 alkenyl, C2-10 alkynyl, each of which is independently optionally
substituted at each
occurrence with one or more substituents selected from halogen, -0R100, -
SR100, _N(R100)2, _
S(0)R' ,
_s(0)2R100, _c(0)R100, _
C(0)0R100, -0C(0)R' , -NO2, =0, =S,
160
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
=N(Rioo),
-P(0)(0R1 ), -0P(0)(ORM)2, -C N, C3-10 carbocycle and 3- to 10-membered
heterocycle; and C3_10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3_10
carbocycle and 3- to 10-membered heterocycle in R6 is optionally substituted
with one or more
substituents selected from halogen, -0Rioo, _sRioo, _N(Rioo)2, _s(0)Rioo,
_s(0)2Rioo _
C(0)R' , _
C(0)0Rioo, -0C(0)R1 , -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1 ), -0P(0)(ORM)2, -C N, C1_6 alkyl, C2lk -6 aenyl, C2_6 alkynyl;
Rm at each occurrence is independently selected from hydrogen; and C1_10
alkyl, C2-10
alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle
each of which is
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, =S, and haloalkyl; and
X3 is a linker moiety, wherein at least one of Ri, R2, R3, R4, Rs, xl, )(2, a
b-1
substituent
and a B2 substituent is -X3.
[0415] In various embodiments, the compound of Formula (II) is represented by
a structure of
Formula (IA):
Xi
0 i
p¨o
Z \
Y 0 B1
0
B2 Ck 2
0 N(R1)2
0_P
/ 0
X2
(IA) ,
or pharmaceutically acceptable salts thereof.
[0416] In various embodiments, the compound of Formula (II) is represented by
a structure of
Formula (JIB):
Xi
0 I
P-0
Z
B1
i-- 0
2 1 c
NH(R )2 0
0------
/P
X2 0 (IIB),
or a pharmaceutically acceptable salt thereof.
161
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
[0417] In various embodiments, B1 and B2 are independently selected from
optionally substituted
purines. B1 and B2 may be each, independently selected from one another,
adenine, guanine, and
derivatives thereof. B1 and B2 may be independently selected from optionally
substituted adenine,
optionally substituted guanine, optionally substituted xanthine, optionally
substituted
hypoxanthine, optionally substituted theobromine, optionally substituted
caffeine, optionally
substituted uric acid, and optionally substituted isoguanine. In a preferred
embodiment, B1 and B2
are independently selected from optionally substituted adenine and optionally
substituted guanine.
[0418] In various embodiments, B1 is substituted by X3 and optionally one or
more additional
substituents independently selected from halogen, -ORloo, _swoo, _N(Rioo)2, _
S(0)R1 , -
S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)R' , -NO2, =0, =s, =N(Rioo), _
CN, and R6. For
--X3
H N
1\136\1
N N
example, B1 may be represented by: ..1., , and wherein B1 is optionally
further substituted
by one or more substituents.
[0419] In various embodiments, B2 is substituted by X3 and optionally one or
more additional
substituents independently selected from halogen, -ORloo, _swoo, _N(Rioo)2, _
S(0)R1 , -
S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)R' , -NO2, =0, =s, =N(Rioo), _
CN, and R6. For
--X3
H N
1\136\1
N N
example, B2 may be represented by: ¨1¨ , and wherein B2 is optionally
further substituted
by one or more substituents.
--X3
H N
1\136\1
N N
[0420] In some embodiments, B1 is represented by 'AL and B2 is represented
by
H N X3
NH2
N
1\1X(1
261
N N N N
In some embodiments, B1 is represented by ..1.. and
B2 is represented by
0
ND.(NH
I
N N NH2
.
162
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0421] In various embodiments, Xl is selected from -0- X3 and-S-X3. In some
embodiments, Xl
is selected from ¨OH and ¨SH. In some embodiments, Xl is -SH.
[0422] In various embodiments, X2 is selected from -0- X3 and-S-X3. In some
embodiments, X2
is selected from ¨OH and ¨SH. In some embodiments, X2 is -S-X3.
[0423] In some embodiments, Xl is ¨SH and X2 is -S-X3.
[0424] In certain embodiments, Y is selected from -NR4X3, -S-X3, and -0- X3.
In some
embodiments, Y is selected from ¨OH, -SH, ¨0-Ci_io alkyl, ¨NH(Ci_io alkyl),
and ¨NH2. In a
preferred embodiment, Y is selected from ¨OH.
[0425] In various embodiments, Z is selected from -NR4X3, -S-X3, and -0- X3.
In some
embodiments, Z is selected from ¨OH, -SH, ¨0-Ci_io alkyl, ¨NH(Ci_io alkyl),
and ¨NH2.
[0426] In various embodiments, ¨X3 is represented by the formula:
J'L
' 0
N peptide
[0427] In some embodiments, ¨X3 is represented by the formula:
N peptide¨RX
, wherein RX comprises a reactive moiety, such a maleimide.
[0428] In some embodiments, ¨X3 is represented by the formula:
0 J(
N peptide¨RX"¨Antibody
, wherein RX* is a reactive moiety that has reacted
with a moiety on an antibody construct to form a conjugate.
[0429] In some embodiments, ¨X3 is represented by the formula:
µz)(=.( ONJ(/RX
0 0
/1-4 H , wherein RX is a reactive moiety, such as
a maleimide.
[0430] In some embodiments, ¨X3 is represented by the formula:
0 0
RX* Antibody
1-4 H , wherein RX* is a reactive
moiety that has reacted with a moiety on an antibody construct to form a
conjugate, such as an
antibody drug conjugate.
163
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0431] In some embodiments, the compound is represented by the formula:
o
<i\I fNLH
x19 N N NH2
HO
0
"----0L 1,C77?iN0 H
0 0
0-P, X
H2N,T;NrIN
0 0 0
HN I 0
0 ,
or a pharmaceutically acceptable salt thereof. The compound may be represented
by the formula:
N NH
<1 I
.1 ( i p N NE42
-
H
0 OR HN-Tr\(::,.
H2N N \1.?
0 OH 0 0
HZIP 0
N
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
<,N 2 1 \ 11,H
HS N N NF
H
HN,ir-000,oN
H2N ,11 -Ek
0
0 SH 0
V 1)Nl'j I \ 0 0
N
O , or a pharmaceutically
acceptable salt thereof.
[0432] In some embodiments, the compound is represented by the formula:
o
NINH
1 ,I,
)0P N N NH2
FIC4 Ci
H C1-135Fi
H2N 0
V,y.wwil
0-F;2 HN 0 =N NN
0 H 0
HN I
o,...)
N , d x
0
11
N
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
NINH
1
HOP N N NH2
r P-0
Hiy_zp cL0_1?
H cH3H
ro--L A HN 40 1\11('N Ir..).1...3
H2N)NrzN 0-El 0 =0 H 0
0 OH
HN; I 1> 0 0
N
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
164
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
111)1 *LNH
HS. P N N NH2
H r P-0
I,Lzp :;_,
H cH3j5....Ei
nCri =r HN io kilr''N Y.W....3N
H2N);NrI.N 0-Ek 0 0 H 0
o SH
HN I 0 0
N
O , or a pharmaceutically acceptable salt
thereof.
[0433] In some embodiments, the compound is represented by the formula:
o
)(9 N N NH2
OH
Ic_CL? F
0 F
1-121\1);NrIN
0 X 0 0 F
HN I NI> F
F
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
<,N 1,,,,ILH
HOP N N NH2
,P-0
OH F
0_,Fc nN--y--.....0,õ----Ø-.õ-o,õõ--.Ø--
,....,.,0, = 00 F
H2N N N
);Z N /> 0 OH 0 0F F
HN F
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
<pi 1,,III,H
HS. P N N NH2
r P-0
OH F
H2N N N
14-A0_,F(o HN----1(0/0,0,/.r / 0 F
);Z 0 SH 0 0F F
HN N F
O , or a pharmaceutically acceptable
salt thereof.
[0434] In some embodiments, the compound is represented by the formula:
o
1\11), ,LNH H2N,r0
)0P N N NH2 FIN
FIC4 Ci
H
E =-= H 0
,(77-r N,,=,'
H2N,r;N Xr1IN 0-1?µ 2 HN 0 io
0 H 0
0
HN I NI> 0 0
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
165
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
< H2N yo
HOP N N NH2 HN
HOL Thc,L)
0-e i- w 0 H
H2N N N HN 0
0
0 OH
EIV I V> 0 0
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
\ 11-12:,NLH H 2 N yo
HS. P N N NH2 HN
H/ PO Hksi.... I
N '
laH\
H2N;1 0-1 HNr-c)
0 SH
=w 0 H
0
0 0
HN1. N
O , or a pharmaceutically acceptable salt
thereof.
[0435] In some embodiments, the compound is represented by the formula:
0
NX.ILNH
1
xP N N NH2
FLO
O1-417
0
0--r
H2 N N d x2 o o
V NI H 0
N
O , or a pharmaceutically acceptable
salt thereof.
[0436] The compound is represented by the formula:
HOP N N NH2
, P-0
OFikr
H2Nk, NN
00H 0 0
HNYL N/> 0
O , or a pharmaceutically acceptable
salt thereof.
166
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0437] The compound may be represented by the formula:
<,N1
HS. P N N NH2
P-0
OF2Ltir
I ,F(SH 0 0 0
o HN-..rripolp,313.rip-T\
H N N
2 N
0
Nv
H 1>
N
O , or a pharmaceutically acceptable
salt thereof.
[0438] In some embodiments, the compound is represented by the formula:
o o o
H /
0
x1P N N
I-0
HO ,
Ica_
OH
H2N,r1NrIN = 0 / FLX2 -..,
HN I NI> d
o , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
H N jOIC)0 NHj.)1..3
/
N1/LN
1 0
HOP N N
, P-0
7_.._.
O p OH
H2N N NI 0 \ /
)j' µ; P-OH
HN N 0
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
H1\10 0 . il...3N /
NI/IN
' 1 0
HS. P N N
, P-0
O P S H
H2N *NN
N S- OH
H N
O , or a pharmaceutically
acceptable salt thereof.
167
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0439] In some embodiments, the compound is represented by the formula:
o
o o
HN
H
<NX.L.K1 a
1 I
X19 N N
P-0
HO ,
j_ Ji)
(---0A 01-1,0
H2NN N o-----, 7
HNYT.NI>
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
N1/LN
' I 0
HOP N N
, P-0
7___3'L cLC:_i
0 OH ,0
H2N N N 0---_____ /
H
NV N/> NH
O , or a pharmaceutically
actable salt thereof. The compound may be represented by the formula:
N1/LN
' I 0
HS. P N N
, P-0
7___3'L cLC:_i
0 OH ,0
H2N N N
HN; N
)T /> 13 SH
O , or a pharmaceutically
acceptable salt thereof.
[0440] In some embodiments, the compound is represented by the formula:
o o
\ID --,--
HNI--10 io
51 N
H 0
X1P N N
P-0o
1-1C4i7(
OT-17
H2N N 0-p,---2--"
0 X
FiV 1 \S
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
168
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
HITIO
NN
11511 3 H 0
(' I
HOP N N
P-0
7__srL
0 OH
H2Ny:NrzN
6 OH
HN I
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
O o
HN o)Lo 0 ly(r1-1 i ,K,......õ..........
,ii?
Nii N
Nx'L,N N
H 0
HS, P N N
P-0
HO ,
)_.4)
µ-------0 07-1 I
0-P-----"
H2NN N
0 SH
HNYTN/>
O , or a pharmaceutically acceptable
salt thereof.
In some embodiments, the compound is represented by the formula:
O o
HN-A0
N No
N'N
" I H 0 H 0
x19 N N
P-0 HN
HC4Li7(
H2N0
0 077
0-pc---"
H2NN N d x2
HiljN/>
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
....,7 0
HN1.0 io N N H µ õit.õ.....,....õõ..... I'LNI Kilr'H
N
0
0
HOP N N H
HN
H2N 0
0 OH
0_,---0
H21\y,;IN 0 OH
HN I
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
169
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0
HN 0 110 çL
H 0 H 0
HS.
P-0 HN
Hy_ jfir IcLCL?
H2N 0
0
H N N
2 FIVN 6 SH
0 ,
or a pharmaceutically acceptable
salt thereof.
[0441] In some embodiments, the immune-stimulatory compound is a damage-
associated
molecular pattern molecule or a pathogen-associated molecular pattern
molecule.
[0442] In some embodiments, the immune-stimulatory compound is a Toll-like
receptor agonist,
STING agonist, or RIG-I agonist.
[0443] In some embodiments, the immune-stimulatory compound is a CpG
oligonucleotide,
Poly G10, Poly G3, Poly I:C, Lipopolysaccharide, zymosan, flagellin, Pam3CSK4,
PamCysPamSK4, dsRNA, a diacylated lipopeptide, a triacylated lipoprotein,
lipoteichoic acid, a
peptidoglycan, a cyclic dinucleotide, a 5'ppp-dsRNA, S-27609, CL307, UC-IV150,
imiquimod,
gardiquimod, resiquimod, motolimod, VTS-1463G5-9620, G5K2245035, TMX-101, TMX-
201,
TMX-202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-
34240,
KU34B, 5B9200, SB11285, 8-substituted imidazo[1,5-a]pyridine, or CL663.
[0444] In some embodiments, the immune-stimulatory compound is an inhibitor of
TGFB, Beta-
Catenin, PI3K-beta, STAT3, IL-10, IDO, or TDO. In some embodiments, the immune-
stimulatory compound is LY2109761, G5K263771, iCRT3, iCRT5, iCRT14, LY2090314,
CGX-
1321, PRI-724, BC21, ZINCO2092166, LGK974, IWP2, LY3022859, LY364947,
SB431542,
AZD8186, SD-208, indoximod (NLG8189), F001287, GDC-0919, epacadostat
(INCB024360),
RG70099, 1-methyl-L-tryptophan, methylthiohydantoin tryptophan, bras sin,
annulin B,
exiguamine A, PIM, LM10, 8-substituted 2-amino-3H-benzo[b]azepine-4-
carboxamide, or
INCB023843.
[0445] In some embodiments, the immune-stimulatory compound does not reduce
the affinity of
the recombinant bispecific antibody for binding to the tumor associated
antigen or to the antigen
on the antigen presenting cell.
Chemotherapeutic Agent Recombinant Bispecific Antibody Conjugates
[0446] In certain embodiments, the recombinant bispecific antibodies further
comprise a
chemotherapeutic compound. The recombinant bispecific antibody further
comprising a
chemotherapeutic compound can be a recombinant bispecific antibody conjugate.
The
chemotherapeutic compound can be coupled to the Fc region of the recombinant
bispecific
170
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
antibody. In certain embodiments, the chemotherapeutic compound is covalently
coupled to the
recombinant bispecific antibody by a linker creating a recombinant bispecific
antibody conjugate.
In certain embodiments, the coupled chemotherapeutic compound comprises an
alkylating agent
(e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,
mechlorethamine,
uramustine, thiotepa, nitrosoureas, or temozolomide), an anthracycline (e.g.,
doxorubicin,
adriamycin, daunorubicin, epirubicin, or mitoxantrone), a cytoskeletal
disruptor (e.g., paclitaxel
or docetaxel), a histone deacetylase inhibitor (e.g., vorinostat or
romidepsin), an inhibitor of
topoisomerase (e.g., irinotecan, topotecan, amsacrine, etoposide, or
teniposide), a kinase inhibitor
(e.g., bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, or
vismodegib), a nucleoside analog
or precursor analog (e.g., azacitidine, azathioprine, capecitabine,
cytarabine, fluorouracil,
gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine), a
peptide antibiotic
(e.g., actinomycin or bleomycin), a platinum-based agent (e.g., cisplatin,
oxaloplatin, or
carboplatin), or a plant alkaloid (e.g., vincristine, vinblastine,
vinorelbine, vindesine,
podophyllotoxin, paclitaxel, or docetaxel). In some embodiments, the
chemotherapeutic agent is
a nucleoside analog. In some embodiments, the chemotherapeutic agent is
gemcitabine. The
chemotherapeutic compound can be coupled to the recombinant bispecific
antibody via a linker,
as further described herein.
Linkers
[0447] The conjugates and methods of using such conjugates described herein
include conjugates
that can comprise a linker, e.g., cleavable or non-cleavable linker, attached
to an antibody
construct or to a recombinant bispecific antibody. Linkers of the conjugates
and methods
described herein may not affect the binding of active portions of a conjugate
(e.g., active portions
include antigen binding domains, Fc domains, Fc comprising domains, binding
domains,
antibodies (e.g., recombinant bispecific antibodies), antibody constructs,
agonists or the like) to a
target, which can be a cognate binding partner such as an antigen. A linker
can form a linkage
between different parts of a conjugate. A conjugate can comprise multiple
linkers. These linkers
can be the same linkers or different linkers. As will be appreciated by the
skilled artisan, the
following description of conjugates comprising antibody constructs is
applicable to conjugates
comprising recombinant bispecific antibodies.
[0448] As will be appreciated by skilled artisans, the linkers can link the
immune-stimulatory
compound to the antibody construct of the conjugate by forming a covalent
linkage to the
immune-stimulatory compound at one location and a covalent linkage to the
antibody construct
of the conjugate at another location. The covalent linkages can be formed by
reaction between
functional groups on the linker and functional groups on the compounds and
antibody construct.
171
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
As used herein, the expression "linker" can include (i) unconjugated forms of
the linker that can
include a functional group capable of covalently linking the linker to an
immune-stimulatory
compound and a functional group capable of covalently linking the linker to an
antibody
construct; (ii) partially conjugated forms of the linker that can include a
functional group capable
of covalently linking the linker to an antibody construct of the conjugate and
that can be
covalently linked to an immune-stimulatory compound, or vice versa; and (iii)
fully conjugated
forms of the linker that can be covalently linked to both an immune-
stimulatory compound and
an antibody construct. In some specific embodiments immune-stimulatory
conjugates described
herein, moieties comprising the functional groups on the linker and covalent
linkages formed
between the linker and antibody construct of the conjugate can be specifically
illustrated as Rx
and LK, respectively. One embodiment pertains to a conjugate formed by
contacting an antibody
construct that binds to a cell surface receptor or tumor antigen expressed on
a tumor cell with a
linker described herein under conditions in which the linker covalently links
to the antibody
construct. One embodiment pertains to a method of making a conjugate formed by
contacting a
linker described herein under conditions in which the linker covalently links
to an antibody
construct. One embodiment pertains to a method of stimulating immune activity
in a cell that
expresses CD40, comprising contacting the cell with a conjugate described
herein that is capable
of binding the cell, under conditions in which the conjugate binds the cell.
[0449] Attachment via a linker can involve incorporation of a linker between
parts of a conjugate.
A linker can be short, flexible, rigid, cleavable, non-cleavable, hydrophilic,
or hydrophobic. A
linker can contain segments that have different characteristics, such as
segments of flexibility or
segments of rigidity. The linker can be chemically stable to extracellular
environments, for
example, chemically stable in the blood stream, or may include linkages that
are not stable. The
linker can include linkages that are designed to cleave and/or immolate or
otherwise breakdown
specifically or non- specifically inside cells. A cleavable linker can be
sensitive to enzymes. A
cleavable linker can be cleaved by enzymes such as proteases. A cleavable
linker can be contain
a valine-citrulline peptide or a valine-alanine peptide. A valine-citrulline-
or valine-alanine-
containing linker can contain a pentafluorophenyl group. A valine-citrulline
or valine-alanine-
containing linker can contain a succimide or a maleimide group. A valine-
citrulline- or valine-
alanine-containing linker can contain a para aminobenzoic acid (PABA) group. A
valine-
citrulline- or valine-alanine-containing linker can contain a PABA group and a
pentafluorophenyl
group. A valine-citrulline- or valine-alanine-containing linker can contain a
PABA group and a
succinimide group. A valine-citrulline- or valine-alanine-containing linker
can contain a PABA
group and a maleimide group. A non-cleavable linker can be protease
insensitive. A non-
172
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cleavable linker can contain a maleimide group. A non-cleavable linker can
contain a
succinimide group. A non-cleavable linker can be maleimidocaproyl linker. A
maleimidocaproyl
linker can comprise N-maleimidomethylcyclohexane-l-carboxylate. A
maleimidocaproyl linker
can contain a succinimide group. A maleimidocaproyl linker can contain
pentafluorophenyl
group. A linker can be a combination of a maleimidocaproyl group and one or
more polyethylene
glycol molecules. A linker can be a maleimide-PEG4 linker. A linker can be a
combination of a
maleimidocaproyl linker containing a succinimide group and one or more
polyethylene glycol
molecules. A linker can be a combination of a maleimidocaproyl linker
containing a
pentafluorophenyl group and one or more polyethylene glycol molecules. A
linker can contain
maleimides linked to polyethylene glycol molecules in which the polyethylene
glycol can allow
for more linker flexibility or can be used lengthen the linker. A linker can
be a
(maleimidocaproy1)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker. A
linker can be a
THIOMAB linker. A THIOMAB linker can be a (maleimidocaproy1)-(valine-
citrulline)-(para-
aminobenzyloxycarbonyl) linker. A linker can also be an alkylene, alkenylene,
alkynylene,
polyether, polyester, polyamide, polyamino acids, polypeptides, cleavable
peptides, or
aminobenzylcarbamates. A linker can contain a maleimide at one end and an N-
hydroxysuccinimidyl ester at the other end. A linker can contain a lysine with
an N-terminal
amine acetylated, and a valine-citrulline cleavage site. A linker can be a
link created by a
microbial transglutaminase, wherein the link can be created between an amine-
containing moiety
and a moiety engineered to contain glutamine as a result of the enzyme
catalyzing a bond
formation between the acyl group of a glutamine side chain and the primary
amine of a lysine
chain. A linker can contain a reactive primary amine. A linker can be a
Sortase A linker. A
Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG
recognition motif (SEQ
ID NO: 672) to an N-terminal GGG motif to regenerate a native amide bond. The
linker created
can therefore link a moiety attached to the LXPTG recognition motif (SEQ ID
NO: 672) with a
moiety attached to the N-terminal GGG motif. A linker can be a link created
between an
unnatural amino acid on one moiety reacting with oxime bond that was formed by
modifying a
ketone group with an alkoxyamine on another moiety. A moiety can be a
conjugate. A moiety
can be an antibody construct, such as an antibody. A moiety can be an immune-
stimulatory
compound. A moiety can be a binding domain. A linker can be unsubstituted or
substituted, for
example, with a substituent. A substituent can include, for example, hydroxyl
groups, amino
groups, nitro groups, cyano groups, azido groups, carboxyl groups,
carboxaldehyde groups,
imine groups, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups,
acyl groups, acyloxy
groups, amide groups, and ester groups.
173
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0450] In a conjugate as described herein, the immune-stimulatory compound can
be linked to
the antibody construct of the conjugate by way of linkers. The linker linking
an immune-
stimulatory compound to the antibody construct of the conjugate can be short,
long, hydrophobic,
hydrophilic, flexible or rigid, or may be composed of segments that each
independently have one
or more of the above-mentioned properties such that the linker may include
segments having
different properties. The linkers can be polyvalent such that they covalently
link more than one
immune-stimulatory compound to a single site on the antibody construct, or
monovalent such
that covalently they link a single immune-stimulatory compound to a single
site on the antibody
construct of the the conjugate.
[0451] Exemplary polyvalent linkers that may be used to link many immune-
stimulatory
compounds to an antibody construct of the conjugate are described. For
example, Fleximer
linker technology has the potential to enable high-DAR conjugate with good
physicochemical
properties. As shown below, the Fleximer linker technology is based on
incorporating drug
molecules into a solubilizing poly-acetal backbone via a sequence of ester
bonds. The
methodology renders highly-loaded conjugates (DAR up to 20) whilst maintaining
good
physicochemical properties. This methodology could be utilized with immune-
stimulatory
compound as shown in the Scheme below.
X1 0 iX1
0 1
p¨
P¨
B1 \
7 \ y 0/ B1
y 0
0
0
2
B2 ok 0 HN
0 HN
)ri
0-1:3 0¨p
/o
/ 0 OH X2 0
X2
01_
NH2
174
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0 - =
ack
U ^c.,,yrOya Thr6ya
g
Ho OH OH n
Ho
F.'eximer4nker
INF'
HN HN HN
0 0111,K
b-Drug" 0-Dwg' 0-Drug'
[0452] To utilize the Fleximer linker technology depicted in the scheme
above, an aliphatic
alcohol can be present or introduced into the immune-stimulatory compound. The
alcohol moiety
is then conjugated to an alanine moiety, which is then synthetically
incorporated into the
Fleximer linker. Liposomal processing of the conjugate in vitro releases the
parent alcohol-
containing drug.
[0453] By way of example and not limitation, some cleavable and noncleavable
linkers that may
be included in the conjugates described herein are described below.
[0454] Cleavable linkers can be cleavable in vitro and in vivo. Cleavable
linkers can include
chemically or enzymatically unstable or degradable linkages. Cleavable linkers
can rely on
processes inside the cell to liberate an immune-stimulatory compound, such as
reduction in the
cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by
specific proteases or
other enzymes within the cell. Cleavable linkers can incorporate one or more
chemical bonds that
are either chemically or enzymatically cleavable while the remainder of the
linker can be non-
cleavable.
[0455] A linker can contain a chemically labile group such as hydrazone and/or
disulfide groups.
Linkers comprising chemically labile groups can exploit differential
properties between the
plasma and some cytoplasmic compartments. The intracellular conditions that
can facilitate
immune-stimulatory compound release for hydrazone containing linkers can be
the acidic
environment of endosomes and lysosomes, while the disulfide containing linkers
can be reduced
in the cytosol, which can contain high thiol concentrations, e.g.,
glutathione. The plasma stability
of a linker containing a chemically labile group can be increased by
introducing steric hindrance
using substituents near the chemically labile group.
[0456] Acid-labile groups, such as hydrazone, can remain intact during
systemic circulation in
the blood's neutral pH environment (pH 7.3-7.5) and can undergo hydrolysis and
can release the
immune-stimulatory compound once the conjugate is internalized into mildly
acidic endosomal
175
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
(pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of the cell. This pH
dependent release
mechanism can be associated with nonspecific release of the drug. To increase
the stability of the
hydrazone group of the linker, the linker can be varied by chemical
modification, e.g.,
substitution, allowing tuning to achieve more efficient release in the
lysosome with a minimized
loss in circulation.
[0457] Hydrazone-containing linkers can contain additional cleavage sites,
such as additional
acid-labile cleavage sites and/or enzymatically labile cleavage sites.
Conjugates including
exemplary hydrazone-containing linkers can include, for example, the following
structures:
n
N" N -Ab
(Is)
D)
0 -
N A
Oh) N
0
Cs;
D,N
tli)
H
0
0 Jrt
wherein D and Ab represent the immune-stimulatory compound and antibody
construct,
respectively, and n represents the number of immune-stimulatory compound -
linkers linked to
the antibody construct. In certain linkers such as linker (Ig), the linker can
comprise two
cleavable groups¨ a disulfide and a hydrazone moiety. For such linkers,
effective release of the
unmodified free immune-stimulatory compound can require acidic pH or disulfide
reduction and
acidic pH. Linkers such as (Ih) and (Ii) can be effective with a single
hydrazone cleavage site.
[0458] Other acid-labile groups that can be included in linkers include cis-
aconityl-containing
linkers. cis-Aconityl chemistry can use a carboxylic acid juxtaposed to an
amide bond to
accelerate amide hydrolysis under acidic conditions.
176
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0459] Cleavable linkers can also include a disulfide group. Disulfides can be
thermodynamically stable at physiological pH and can be designed to release
the immune-
stimulatory compound upon internalization inside cells, wherein the cytosol
can provide a
significantly more reducing environment compared to the extracellular
environment. Scission of
disulfide bonds can require the presence of a cytoplasmic thiol cofactor, such
as (reduced)
glutathione (GSH), such that disulfide-containing linkers can be reasonably
stable in circulation,
selectively releasing the immune-stimulatory compound in the cytosol. The
intracellular enzyme
protein disulfide isomerase, or similar enzymes capable of cleaving disulfide
bonds, can also
contribute to the preferential cleavage of disulfide bonds inside cells. GSH
can be present in cells
in the concentration range of 0.5-10 mM compared with a significantly lower
concentration of
GSH or cysteine, the most abundant low-molecular weight thiol, in circulation
at approximately
tM. Tumor cells, where irregular blood flow can lead to a hypoxic state, can
result in enhanced
activity of reductive enzymes and therefore even higher glutathione
concentrations. The in vivo
stability of a disulfide-containing linker can be enhanced by chemical
modification of the linker,
e.g., use of steric hindrance adjacent to the disulfide bond.
[0460] Conjugates including exemplary disulfide-containing linkers can include
the following
structures:
R R
(1:1)
R R 0
(1k) ,s
, n
R
(n) Ab
wherein D and Ab represent the immune-stimulatory compound and antibody
construct,
respectively, n represents the number of immune-stimulatory compound-linkers
linked to the
antibody construct and R is independently selected at each occurrence from
hydrogen or alkyl,
for example. Increasing steric hindrance adjacent to the disulfide bond can
increase the stability
177
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
of the linker. Structures such as (Ij) and (I1) can show increased in vivo
stability when one or
more R groups is selected from a lower alkyl such as methyl.
[0461] Another type of linker that can be used is a linker that is
specifically cleaved by an
enzyme. For example, the linker can be cleaved by a lysosomal enzyme. Such
linkers can be
peptide-based or can include peptidic regions that can act as substrates for
enzymes. Peptide
based linkers can be more stable in plasma and extracellular milieu than
chemically labile linkers.
[0462] Peptide bonds can have good serum stability, as lysosomal proteolytic
enzymes can have
very low activity in blood due to endogenous inhibitors and the unfavorably
high pH value of
blood compared to lysosomes. Release of an immune-stimulatory compound from a
conjugate
can occur due to the action of lysosomal proteases, e.g., cathepsin and
plasmin. These proteases
can be present at elevated levels in certain tumor tissues. The linker can be
cleavable by a
lysosomal enzyme. The lysosomal enzyme can be, for example, cathepsin B, P-
glucuronidase, or
P-galactosidase.
[0463] In a linker, a cleavable peptide can be selected from tetrapeptides
such as Gly-Phe-Leu-
Gly (SEQ ID NO: 1332), Ala-Leu-Ala-Leu (SEQ ID NO: 1333) or dipeptides such as
Val-Cit,
Val-Ala, and Phe-Lys. Dipeptides can have lower hydrophobicity compared to
longer peptides,
depending on the composition of the peptide.
[0464] A variety of dipeptide-based cleavable linkers can be used in the
conjugates described
herein.
[0465] Enzymatically cleavable linkers can include a self-immolative spacer to
spatially separate
the immune-stimulatory compound from the site of enzymatic cleavage. The
direct attachment of
an immune-stimulatory compound to a peptide linker can result in proteolytic
release of an amino
acid adduct of the immune-stimulatory compound, thereby impairing its
activity. The use of a
self-immolative spacer can allow for the elimination of the fully active,
chemically unmodified
immune-stimulatory compound upon amide bond hydrolysis.
[0466] One self-immolative spacer can be a bifunctional para-aminobenzyl
alcohol group, which
can link to the peptide through the amino group, forming an amide bond, while
amine containing
immune-stimulatory compounds can be attached through carbamate functionalities
to the
benzylic hydroxyl group of the linker (to give a p-amidobenzylcarbamate,
PABC). The resulting
pro-immune-stimulatory compound can be activated upon protease-mediated
cleavage, leading to
a 1,6-elimination reaction releasing the unmodified immune-stimulatory
compound, carbon
dioxide, and remnants of the linker group. The following scheme depicts the
fragmentation of p-
amidobenzyl carbamate and release of the immune-stimulatory compound:
178
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
0 r
4,.. ,..t.-
c-'-===
0 7) 0 X.--1) __ protease* i ..,:-....\-.1"Ncy' \247D
i 1,6-eliT!ne,11:iol, 1 i
r
..... ,,,,,.......,, +c02
.,
wherein X-D represents the unmodified immune-stimulatory compound.
[0467] The enzymatically cleavable linker can be a 13-glucuronic acid-based
linker. Facile release
of the immune-stimulatory compound can be realized through cleavage of the 13-
glucuronide
glycosidic bond by the lysosomal enzyme 13-glucuronidase. This enzyme can be
abundantly
present within lysosomes and can be overexpressed in some tumor types, while
the enzyme
activity outside cells can be low. 13- Glucuronic acid-based linkers can be
used to circumvent the
tendency of a conjugate to undergo aggregation due to the hydrophilic nature
of13-glucuronides.
In certain embodiments, 13-glucuronic acid-based linkers can link an antibody
construct to a
hydrophobic immune-stimulatory compound. The following scheme depicts the
release of an
immune-stimulatory compound (D) from an antibody construct of the conjugate
(Ab) containing
a 13-glucuronic acid-based linker:
HO
HO¨ .,
HO.1 V 0
...,,,_ 0
s Y -;.----="'`O`KO fl-Ot.kotmWaw
HO
....
'' ikr=
N
`k ii, N. = .fmµ:::rmrtaton -
-t*CO2
.
) HN ---.., HN ..,=.,
HsLy-''s.,kb H,e k . , An
D y Ab
8
ofi
[0468] A variety of cleavable P-glucuronic acid-based linkers useful for
linking drugs such as
auristatins, camptothecin and doxorubicin analogues, CBI minor-groove binders,
and psymberin
to antibodies have been described. All of these P-glucuronic acid-based
linkers may be used in
the conjugates comprising an immune-stimulatory compound described herein. In
certain
embodiments, the enzymatically cleavable linker is a P-galactoside-based
linker. P-Galactoside is
present abundantly within lysosomes, while the enzyme activity outside cells
is low.
[0469] Additionally, immune-stimulatory compounds containing a phenol group
can be
covalently bonded to a linker through the phenolic oxygen. One such linker
relies on a
methodology in which a diamino-ethane "Space Link" is used in conjunction with
traditional
"PABO" -based self-immolative groups to deliver phenols.
179
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0470] Immune-stimulatory compounds containing an aromatic or aliphatic
hydroxyl group can
be covalently bonded to a linker through the hydroxyl group using a
methodology that relies on a
methylene carbamate linkage, as described in WO 2015/095755.
[0471] Cleavable linkers can include non-cleavable portions or segments,
and/or cleavable
segments or portions can be included in an otherwise non-cleavable linker to
render it cleavable.
By way of example only, polyethylene glycol (PEG) and related polymers can
include cleavable
groups in the polymer backbone. For example, a polyethylene glycol or polymer
linker can
include one or more cleavable groups such as a disulfide, a hydrazone or a
dipeptide.
[0472] Other degradable linkages that can be included in linkers can include
ester linkages
formed by the reaction of PEG carboxylic acids or activated PEG carboxylic
acids with alcohol
groups on an immune-stimulatory compound, wherein such ester groups can
hydrolyze under
physiological conditions to release the immune-stimulatory compound.
Hydrolytically
degradable linkages can include, but are not limited to, carbonate linkages;
imine linkages
resulting from reaction of an amine and an aldehyde; phosphate ester linkages
formed by reacting
an alcohol with a phosphate group; acetal linkages that are the reaction
product of an aldehyde
and an alcohol; orthoester linkages that are the reaction product of a formate
and an alcohol; and
oligonucleotide linkages formed by a phosphoramidite group, including but not
limited to, at the
end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
[0473] A linker can comprise an enzymatically cleavable peptide moiety, for
example, a linker
comprising structural formula (IVa), (IVb), (IVc), or (IVd):
180
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
$
tiVa) N
likbN 'T PePticte.¨N"
H
Y
RY 0
y
(I V13) 0
-
4 p pepticte¨N
0
0
(TV)
-jLLpeptkie¨N
RY 0
Q
(1Vd) 1!4
peptde¨N'
or a salt thereof, wherein: peptide represents a peptide (illustrated N¨>C,
wherein peptide
includes the amino and carboxy "termini") a cleavable by a lysosomal enzyme; T
represents a
polymer comprising one or more ethylene glycol units or an alkylene chain, or
combinations
thereof; Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate;
RY is hydrogen or
Ci_4 alkyl-(0),-(C14 a1kylene)s-G1 or Ci_4 alkyl-(N)-[(C1-4 alkylene)-02; Rz
is C1-4 alkyl-(0)r-
(C14 a1kylene)s-G2; is SO3H, CO2H, PEG 4-32, or sugar moiety; G2 is SO3H,
CO2H, or PEG
4-32 moiety; r is 0 or 1; s is 0 or 1; p is an integer ranging from 0 to 5; q
is 0 or 1; xis 0 or 1; y is
0 or 1; represents the point of attachment of the linker to the immune-
stimulatory compound;
and * represents the point of attachment to the remainder of the linker.
181
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0474] In certain embodiments, the peptide can be selected from a tripeptide
or a dipeptide. In
particular embodiments, the dipeptide can be selected from: Val-Cit; Cit-Val;
Ala-Ala; Ala-Cit;
Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-
Cit; Cit-Lys; Asp-Cit;
Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-
Ala; Phe-Cit;
Cit-Phe; Leu- Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and
Trp-Cit, or salts
thereof.
[0475] Exemplary embodiments of linkers according to structural formula (IVa)
that can be
included in the conjugates described herein can include the linkers
illustrated below (as
illustrated, the linkers include a group suitable for covalently linking the
linker in a conjugate and
the wavy line or unlinked bond indicates an attachment site for an immune-
stimulatory
compound):
0 0
11 ;i:11
(liVa 1 ) 0 -)
iv
iN
0
0 0 9
Via.2)
!1 ViI ii
(.)
.Ns - 0
(IV a 3) -
SO:1H
0
.,11F
0
(IVa 4)
-"
1, H
182
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0 0 0
H A
(Bra
H
0 ,.,-
Nt
N
i
0
Ikf.N
4'
(Illaõ7) [1
o
rr
NFL
\siti..A7=0
0.1õ NH2
1.õ
N N
0 8
0
[0476] Exemplary embodiments of linkers according to structural formula (IVb),
(IVc), or (IVd)
that can be included in the conjugates described herein can include the
linkers illustrated below
(as illustrated, the linkers can include a group suitable for covalently
linking the linker to a
conjugate and the wavy line indicates an attachment site for an immune-
stimulatory compound):
183
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
0
1
0 Vb )
1,...-4,. N 4 11 , , , .,..,,
. I ) t i P '
0 .
-0
INH
CP: g
,t)
(1Vb:2) 6 H E H
1
AP
art 0 ( .
I.Vb.3) ici
o
(I VI) . 4)
Ls....r
N4
0 I ;-== 0 ,f-ONisor'''''".ØntC:
L.,..
:
Cf. NHci7
184
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
it 1 (INTb.6)
H a
Viy0
3-44
,
Alb .7)
\L'st
Q
=4iA
04ILNAZ
it
H .11
(V1),8)
(13 IA A
,
0,0"-QH
"
(1Vb9 H
LN
U.
041INN3-12
NI-ks
9 [i
ovb AA)
0
185
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
C:5
A---
0
( . 1) = N
A H H
HO
6
NH
(AN1N2
0
1-
,,
. .14
(ivb.t2) Nr"-
H H
HO-6=0 0
6
NH,
cii
,0
r-11. o
µSr, IZ4
H
(IAA.
0
Ike
0
r.,Lit
(IVb . 4) 0
0
H>:N914.0
186
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
f'...?
k. 1-1 li
" ' IT"'"N''''''N'*''''''t'N '."-e'''' N" = 1.14')11,0:1
(IVb , 15 )
RH
NNsIkb
0
11
H C.'
HO
H '''' = 0
(PI b..17)
0 H -;:õ.,,õ"
m,..../ N
7
(IVb , 1 (0
'3
3 )
:1;
f..'
--:-../
n
ce,bQ
,
-
p
M
187
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
1-----"---k
kmati:., 1,_
.N1--- "40
i
PJ
OH
t
Hee,µ
(, IVb . 19 )
r-t.õ,......
OH
6
o u -
--i
9
, Al
r
N ='.
gY '
(WC , 1 )
1414
EilisNlt:
..,õ..H.,,,,,
0 p
OVC,2) H H
,t,g---1(
*
j L . õ
-
0:4-111
FizN,...e
(1Vc 3 )
e,"-\--.10--44.N.t3., p4.-- ..-'N y".= ett \ ..."-~4
0 fe)
188
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
U... 4) i
0-.1.
0.7.44,.......,
..e. -
(IVe..5)
-6
=
$3,,...0 ',.....Ø-..,..--.0,-
,õØ...,....,.Q,
HQ
(Wc,6) 4*----0 '),......)._
0:1 HN
=
c
9 - ''''' H 0
--iy
\---4\,)-1,---
0 N - -..,-- m --,---
(Pic.7) 0 N 0 E H
Hz.ice-
189
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
.............. 7..--t,='...,.,.e,v-",....."..."`Nve--,,,,,A,..
(Wd, i) ,..,k,.. I--
-:=-1, - ---)
0792Clo
1---
ILIP
r')
. ,,..,,,,,,,
e-....) ,- ,,,----
04 .õ,_ .2) %.. ,
H
(1Vd.3) .,5.4rt. TT: ysi..ty t.y.
c,......,õ,..õ,
=Sr- . .
...Ø--1
0
1-iN)
µ,.
. P LI
(iVii,4) .,...- . ,.N ,,,4¨k
Ci n' )::1 \-R).
..-e-c ,..,, ._ . (...., -
o
Ci
.i=
c'f.v
No
190
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0477] The linker can contain an enzymatically cleavable sugar moiety, for
example, a linker
comprising structural formula (Va), (Vb), (Vc), (Vd), or (Ve):
0 *
..
IN 0
(Va),Ol'i
Q"µ .
rs, ;
'i
OH OH
OH OH
I
(Vb)
$ 0
1-s) .-*
KI
0 X'
- O'''' NP 1 ''''' N.N:N=
=,',1 1
=====. .'''
(VC) ' 0
=kiOH tt, ..,
CY
0H
OH OH
191
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
cH OH
a 6
+2,1
(vd) ---N:s*cy""`h.r=-4----
qil 1
\õ,õ.=..--.:.--,
= :
s.,.H
-se
P *
1
0
zh
r$1/4tN"1-0
(ve) H $ _
0 L'
' 'AH
1
r"'N'Ir OH
OH OH
i
or a salt thereof, wherein: q is 0 or 1; r is 0 or 1; X1 is CH2, 0 or NH; '
represents the point of
attachment of the linker to the immune-stimulatory compound; and * represents
the point of
attachment to the remainder of the linker.
[0478] Exemplary embodiments of linkers according to structural formula (Va)
that may be
included in the immune-stimulatory conjugates described herein can include the
linkers
illustrated below (as illustrated, the linkers include a group suitable for
covalently linking the
linker in a conjugate and the wavy line indicates an attachment site for an
immune-stimulatory
compound):
192
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
S1/2
ere
,,,,N 0
(Va I) 4 ..)1,*
(3
He
-41/41:1, 04k\fits4)
OH
NIA;
(....0
J
(Va:2.) : s "- 9 0 1?
a
, 1
ON
01\
õ
,k
'\' ct
(Va,3)
k,' A
kL, 0 0 NM
Z
tiCr '''tXP
,
NI
R t 1,,,, s
(Va.4.) 0 '..%111.4'W.-'\` "'N'N' '...
.'i
HeeLYt Y\': H
f'd\r)\=)1.4
193
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
I,.k. 0
I NI
CI 0: 0
(Va.3) o
WI' . 'tifi
Oh
k\.,µ,I,
..-,.$
r; `)... 0 0
ke
n
L. )1,,,õ. . AL, =,1
'' µPiN o
..,. i...
Hgtie\7o
N..?
. .0
0 ,.....,k,
z', s*=== l'i 0 0
..
(VA,7) o
1 ,,
1
OdY NOH
OH
r 11
0
0 0
(Va:8) o = ' ''''N' . ''''...\' N'N,--"N.,#"\. -14 s-"e'\,,
H H ,1
hOs 1 1 ....,
1409 1/41.,"'OH
CN
194
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
0 ,...., 0
R , µJ
ffrfTh=-"'sI4.4"H
bH
OiN
d
.."-
.....,
(V a: 1 0)
9
; H H
,-
WILY4'11.
OH
ie,10 :
,
\ $0v-ii 0
(Val 1)
,i1 1 1.3 ty-y-NAk
,\õ::#-,N.----,-----N"--0--- dy
(i?
1 H H
HOe \µy" OH
ati
110.4 0
k, (Va. .12) ,----, 0
a õ.L., o
0
n A H H
KI"Nir 'XIN
Oli
195
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0479] Exemplary embodiments of linkers according to structural formula (Vb)
that may be
included in the conjugates described herein include the linkers illustrated
below (as illustrated,
the linkers include a group suitable for covalently linking the linker in a
conjugate and the wavy
line indicates an attachment site for an immune-stimulatory compound):
0:Att
(Vb. 1 ) o
,
0
ti40204
"
'
(VII 2)
H
0-41c
41¨ D1µµ
H
196
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
.e.''.
A
. Q
)
,---
(Vb,3)
o f
Ziki 6m
Sa* 0....r)=
.6 .,
HNõ.1,01
(Vb,4) i
, .4,, õ.====,....õ...a....,....". -1.'"'". 0
0' t=4 O. =
H L AOH
P
IT4
,....õ,...õ... ti
OH
"
,...:
. ,
0
W....)
--...0,
....Pi
e's,d'Assi" .
..sk 11'''N. = se ,N, '''''s
\\ ..`....
..<- ===-, 0
,f)
0
'....-,..,,,,
197
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
!..K :41M1 0
7
04),7) C-k
,N,s4
,..
ii
om< 4
A
OH
HO 0
*r--"\Notg,
1'.10w \tõ,,..0 =
r,...
,.....,,,,,
-,---N
(vb,8)
2 = =kk fAb__, ,,
S
I
\
I'
Kocs)
:
1 OH
$..
(Vbs9) = 01"\''µ\µ CM
HN
,?=:'0.
\
198
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
.st
0. 0
v
. 1
j,
f
VH=ti i OH
oH o
[0480] Exemplary embodiments of linkers according to structural formula (Vc)
that may be
included in the conjugates described herein include the linkers illustrated
below (as illustrated,
the linkers include a group suitable for covalently linking the linker in a
conjugate and the wavy
line indicates an attachment site for an immune-stimulatory compound):
HO
1= *CS'i
4,3.4t
1:1
(YC, :0: ' e''µ''\''''' =07,,,,
/ ''''''(')
0
."
0
H0
HOft, A
n
k tt ""*C74,
0 4..,'04 0
0 L ""--1(.. -11
, ."s's-1,t '
OIC I)
X2) ( =%1:4.,
\ ,-õ-- k 4
..\ 0
0
.+0
199
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
HO
HaAkels,,,,AOH
L, Os C0.2N,
k 0
(VC.3) 1 N'i 0
1 14 li
0
0+)
N.'kks ...., 0
HO
ks, pH
H(µ)==
L.
I,
0
k µ
(vc, 4) ?z,,:s=AA .)-1 #
s,õ .= '" Nµ b 0
HOS
\
...
6
k,
NA'*'410
2,
9
if 'N , t
:: ,, (
40,. ,, , .,, .
) 1-1
..,..,-.-
0
Ho, ..,....0y6
,
M
õ,,,,,,. ae= NN'i
1 ~
0-H
200
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
04õ,..
0
dt
......s4
= (:::$,,.
e \,...,0 oõ),........
st
o
(V0,6)
0
k ;
0',4
0,
0 ,----%
..,
$..., :
0 1
(Vc
7)
,..,e0Ns.o,,,õ.*AH SO1H < 1 .s
a
1
sok , .
ke oii
OH
Ho
t, 00 f 1
i
0 1 i
( Ve:8) ."µ" .=
A .,,,sk 0
õõI
i
0
201
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
P TA
d 1
(V e.9)
q
q :õ,st
1
c(
L ,
(k:10)
..õ5:::$1i
0 : 0 N"---\
c ..k....A, s
,
.,
,.. , NI N'. \tk
1 H
6H OH
HO
0
Ha'c:
AteLsTie\N"µ -,,,m
a
Wei i)
,
k...
n) (I? oH
A-
[0481] Exemplary embodiments of linkers according to structural formula (Vd)
that may be
included in the conjugates described herein include the linkers illustrated
below (as illustrated,
the linkers include a group suitable for covalently linking the linker in a
conjugate and the wavy
line indicates an attachment site for an immune-stimulatory compound):
202
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
AP
...,,..,t$ ..$..q
-=;.{ --4'.. 0
8 LA"
(Vd, 0 RO Pi 11*
)
µ,)$,K) =.)i.1
(ed,2)
:
KS
0
s:;=., ...... = 0\,..._\
(Vii3)
=,......1 b =3 = )....õ....s. :TN,
=Ne....
HO
0
k "===44
43 0..4
WW1 ....\ .........w...µõN
(Vd ,41) . ...." \
it
0¨ist)
frt6
203
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
õo
r`r
c N.
,i Isito
o
=,iN
(V43)
-\--\
qw- *45.._tai
...ld
N
?
........,,õa.,,,.....,,...,"õeckõ...õ..v....,,,,.................õ,,A
õ."
(Vd,6)
-rm
o' =.M
[0482] Exemplary embodiments of linkers according to structural formula (Ve)
that may be
included in the immune-stimulatory conjugates described herein include the
linkers illustrated
below (as illustrated, the linkers include a group suitable for covalently
linking the linker in a
conjugate and the wavy line indicates an attachment site for an immune-
stimulatory compound):
0
0
1
5.. 11 Pi
OF
c4r1t
..--0
,i.
Z. : firõ, 3,,,,, H
1
'N. ri a.,...,0 ,A.v.....-..,õ0,õ--
,,,,,),,,,,o..,,,--,0,-....õ,, ,, 6 Y
i
1 õxi
1,417:, H
No-
0 sc.:rEs4 0
204
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0483] Although cleavable linkers can provide certain advantages, the linkers
comprising the
conjugate described herein need not be cleavable. For non-cleavable linkers,
the immune-
stimulatory compound release may not depend on the differential properties
between the plasma
and some cytoplasmic compartments. The release of the immune-stimulatory
compound can
occur after internalization of the conjugate via antigen-mediated endocytosis
and delivery to
lysosomal compartment, where the conjugate can be degraded to the level of
amino acids through
intracellular proteolytic degradation. This process can release an immune-
stimulatory compound
derivative, which is formed by the immune-stimulatory compound, the linker, or
a portion
thereof, and in some instances the amino acid residue to which the linker was
covalently attached.
The immune-stimulatory compound derivative from conjugates with non-cleavable
linkers can be
more hydrophilic and less membrane permeable, which can lead to less bystander
effects and less
nonspecific toxicities compared to conjugates with a cleavable linker.
Conjugates with non-
cleavable linkers can have greater stability in circulation than conjugates
with cleavable linkers.
Non-cleavable linkers can be alkylene chains, or can be polymeric, such as,
for example, based
upon polyalkylene glycol polymers, amide polymers, or can include segments of
alkylene chains,
polyalkylene glycols and/or amide polymers. The linker can contain a
polyethylene glycol
segment having from 1 to 6 ethylene glycol units.
[0484] The linker can be non-cleavable in vivo, for example, a linker
according to the
formulations below:
205
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0 0
t 0 t4
ti (149
0
(Vib)
o
0
(WO N
H C14
%
(Vid) ReN.
Ra
or salts thereof, wherein: Ra is selected from hydrogen, alkyl, sulfonate and
methyl sulfonate; 12'
is a moiety including a functional group capable of covalently linking the
linker to an antibody
construct of the conjugate; and represents the point of attachment of the
linker to the
immune-stimulatory compound.
[0485] Exemplary embodiments of linkers according to structural formula (VIa)-
(VId) that may
be included in the conjugates described herein include the linkers illustrated
below (as illustrated,
the linkers include a group suitable for covalently linking the linker in a
conjugate, and
represents the point of attachment in a conjugate):
206
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
91 p 0
q
Nikk,.,,,t ,e''''=,..,1(),N.õ,,,,s\-0-"'\N....--. Nk,,,,--'.\\ wik.,..,--.'N-
N..-4
ci.'
P
, ii
6
0 H
,..,. ,
(Vic:2) , -. = N ...,,,,c.
(:)
o
9 ).....õ...µ
(VW. I)
b
0
9
(VU.. :*\,,,,---\\\,..--"N.,=-N -1(
tOH b
0 0
0 ..,...õõ
(V113) 0
0....
0
(vid.4)
k:k
SO3H 0
207
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0486] Attachment groups that are used to attach the linkers in a conjugate
can be electrophilic in
nature and include, for example, maleimide groups, activated disulfides,
active esters such as
NHS esters and HOBt esters, haloformates, acid halides, alkyl, and benzyl
halides such as
haloacetamides. There are also emerging technologies related to "self-
stabilizing" maleimides
and "bridging disulfides" that can be used in accordance with the disclosure.
[0487] One example of a "self-stabilizing" maleimide group that hydrolyzes
spontaneously under
conjugation conditions to give a conjugate species with improved stability is
depicted in the
schematic below. Thus, the maleimide attachment group is reacted with a
sulfhydryl of a
antibody construct to give an intermediate succinimide ring. The hydrolyzed
(open ring) form of
the attachment group is resistant to deconjugation in the presence of plasma
proteins.
Norms sr-ri: 0
.*----NN
jr¨
')LT--j(
i
/3rN ¨1
-1,;11ALTS C;
.LIU, /¨/
fk-V,11r, Pk.S12. Silla
R
',--4-4 ________________________________________ ¨ 0
--'t o f---). Pro,s. JI., '
_- .-----j
L,µ,
'-- -: As 0
Loads o 11Airl lost' ow 'rime
S.e.t,Ffis9rzOlt.m aita,7hmerti
,-- _
0 0$1
rA ___.cy.,--* FriAL-$H ---.LA .y4A. ,,,,,i.,g,,,,.. ,:t ''-
:1õ,,,,A, "----Km B----- \ --4 = l'ik.
4 =N /.4 Iti. _____ w ,4 ; HN--1 ,
L'.
P017.4
' 4 )=
r...+ Fi2rti HN.
t...
COMM% MSIWNide .omitrirrg wocgfriftqm ¨
:log rtg rl!tdIy fscioclott ide r N
hysko - bvi rarro.. x.v. .ttql- is plosmq
[0488] A method for bridging a pair of sulfhydryl groups derived from
reduction of a native
hinge disulfide bond has been disclosed and is depicted in the schematic
below. An advantage of
this methodology can be the ability to synthesize homogenous DAR4 conjugates
by full
reduction of IgGs (to give 4 pairs of sulfhydryls) followed by reaction with 4
equivalents of the
alkylating agent. Conjugates containing "bridged disulfides" can also have
increased stability.
208
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
,.............,_.N,
d------s---.0
r
.TWkir.:e a.lulfbl.
,. ,-' .,
:
0¨;.71 ,?-i0-0 k
f
,,
=
p'
9 9
n
Glirroatcq
(...µ . 1 Ac.f.:;1.....$,,..; ,, = ,b, .. ).....,(C:
: H
.-
9,1
6
.....,)ogvki
[0489] Similarly, as depicted below, a maleimide derivative that can bridge a
pair of sulfhydryl
groups has been developed.
hrTh.
N.,,.,i
T 0
...14,.. ,
A ,,,
6. ...,
)..."..\(N-k- __________ / S ''k_ dp
1 Q
---- N ,t
0;
[0490] The attachment moiety can contain the following structural formulas
(VIIa), (VIIb), or
(VIIc):
209
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
rr 9
v tila) 0
0\
Rq '10
e kX
A
3,f/1.1170 I 0
N
rst
Gµ
t 0 0
(Vlic) .0
0 N
or salts thereof, wherein: Rq is H or ¨0-(CH2CH20)ii-CH3; x is 0 or 1; y is 0
or 1; G2 is¨
CH2CH2CH2S03H or¨CH2CH20-(CH2CH20)ii-CH3; is¨O-CH2CH2S03H or¨NH(C0)-
CH2CH20-(CH2CH20)12-CH3; and * represents the point of attachment to the
remainder of the
linker.
[0491] Exemplary embodiments of linkers according to structural formula (VIIa)
and (VIIb) that
can be included in the conjugates described herein can include the linkers
illustrated below (as
illustrated, the linkers can include a group suitable for covalently linking
the linker in a conjugate
and the wavy line or unlinked bond indicates an attachment site for an immune-
stimulatory
compound):
210
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
\) sL =====%tt '4%.1".
(V HA .1) = $..,N ' ,=-=
$ ,4,
\ 4t
$.10
I-,
.1\---=
.etif:.........i
c
(V Hal)
i
i r\
r--0 .. 1
= .,,,,,=-
' ' ,.....*
0**.4. J I
L
0..."'`
..x.--.Kv."....j.,
'
=
IN.
(Ali la .3 ) .,
X. '4
t
)(
g.44/C714II tsk
211
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
e
tr
ee
jje
,,,J
j
e
tA
:
1
(V.1las4) r6
f
. .
ccke. tlyki,:(
õt ..--r
6. Noi,,f0
(Vlib. 1 )
1
\,... )
o 'k 4)--
m,
miN 0 ,
. sle'sNiti
N
0 =
,..,'
j
.k,' =-% " 0 ,.....-.N. ...--\,.,..õ..Ø,
õ,....-\\., õ,..-=\ ,0
raeN'sk." N\' 0' `= 0 \'"'
N
tVi1bs2) ( NH
.4:..
4, 4
6 H
...õ,
212
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
q=-1
Ow. i.,...,õ--==="=.1
0 K..
Sss. e
H i 9 H
(Vilb , 3) Ate C.,,*õ.=-= `k's\k,,,,ex ,,,--' :... õ. N. ,..,
i 1
= 0 ===4". \..,,,,,o,,.,
rk ,,,,,,..),,,,H
,,,,,...4
: 4 ....,
OH 64
9H
Or.:S,,,,,,,,,==''''\\,,
:.
k.....
, N ,
H 7 0 H
,..,,,. N ,..).õ,,,... 1 õ....
, A),
(Vilb.4 ) i t
$,T.N.,,,,õ.,..- , 0 ' ...= 0 = ' '
w--
6
a ,=k 14
..,--
....01-,,,,..
CM ON
i
eN
14
0 \I
N=¨== kl
CV Ilb , 6 ) H
#
N=Nr=-;",<N,...- ,Ns=¨"'%õ, 0
.,
,
.,...
.....:
\
...., ,
'"*".NN=rL".' '34
&'=)11 6H
213
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
lib. 7) H
"
====
Q. "
44i OH
0
k ,14o
Cr Y tr"
;=== 0
;
\\
Oillb 8)
t =
ILM OH
[0492] Exemplary embodiments of linkers according to structural formula (VIIc)
that can be
included in the conjugates described herein can include the linkers
illustrated below (as
illustrated, the linkers can include a group suitable for covalently linking
the linker in a
0 Q
O.
H St. sitNt
o o 0
0
0 k 0 OH
`.'01*"'"'=.' OH
OH OH
conjugate):
214
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
,)
\
A
)
e
31Lr '
1 =
(Vito:2)
o %
g ' , R ' = - / 4t^fi
...,,, ,., ..., ......,
...ki
ii.
rA ..0
% ..)
dikk
.:ist..4.""Noe'N\koe'aNt.o.-es.\kµ<=,\;A.,"'s.,,,,N't.1
1,=-=4' (1
%,,,,,,..."N. = 0.,õõ,.....",, -.
r
. .' .., .. h
0
:6
% 5
215
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
HAI:00
HN
o 0
(Vile 4)
=
0
os`
KV, om
,
(VI k 3)
o o
0
= -
0
H H
N= \o,õ)
H
0 o8
(Vibe.6) 8 0 NH
,AOH
t
Conjugates
[0493] A conjugate as described herein can comprise an antibody construct or a
recombinant
bispecific antibody and at least one linker connected to an immune-stimulatory
compound. As
will be appreciated by the skilled artisan, the following description of
conjugates comprising
antibody constructs is applicable to conjugates comprising recombinant
bispecific antibodies.
[0494] In some aspects, the present disclosure provides a conjugate comprising
an immune-
stimulatory compound or salt thereof, an antibody construct, and a linker,
wherein the compound
or salt is linked, i.e., covalently bound, to the antibody construct through
the linker. The linker
can be selected from a cleavable or non-cleavable linker. In some embodiments,
the linker is
cleavable. In alternative embodiments, the linker is non-cleavable. Linkers
are further described
216
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
in the present application in the subsequent section, any one of which can be
used to connect an
antibody to a compound described herein.
[0495] In a conjugate, the drug loading is represented by p, the number of
immune-stimulatory
compound-linker molecules per antibody construct, or the number of immune-
stimulatory
compounds per antibody construct, depending on the particular conjugate.
Depending on the
context, p can represent the average number of immune-stimulatory compounds (-
linker)
molecules per antibody construct, also referred to the average drug loading. P
can range from 1 to
20, from 1-50 or from 1-100. In some conjugates, p is preferably from 1 to 8.
In some preferred
embodiments, when p represents the average drug loading, p ranges from about 2
to about 5. In
some embodiments, p is about 2, about 3, about 4, or about 5. The average
number of immune-
stimulatory compounds per antibody construct in a preparation may be
characterized by
conventional means such as mass spectroscopy, HIC, ELISA assay, and HPLC.
[0496] A conjugate can comprise an antibody construct, an immune-stimulatory
compound, and
a linker. A conjugate can comprise an antibody construct, a pattern
recognition receptor (PRR)
agonist, and a linker. A conjugate can comprise an antibody construct, a
pattern-associated
molecular pattern (PAMP) molecule, and a linker. A conjugate can comprise an
antibody
construct, a damage-associated molecular pattern (DAMP) molecule, and a
linker. A conjugate
can comprise an antibody construct, a STING agonist, and a linker. A conjugate
can comprise an
antibody construct, a toll-like receptor agonist molecule, and a linker. A
conjugate can comprise
an antibody construct, imiquimod, and a linker. A conjugate can comprise an
antibody construct,
S-27609, and a linker. A conjugate can comprise an antibody construct, CL307,
and a linker. A
conjugate can comprise an antibody construct, resiquimod, and a linker. A
conjugate can
comprise an antibody construct, gardiquimod, and a linker. A conjugate can
comprise an
antibody construct, UC-IV150, and a linker. A conjugate can comprise an
antibody construct,
KU34B, and a linker. A conjugate can comprise an antibody construct,
motolimod, and a linker.
A conjugate can comprise an antibody construct, VTX-1463, and a linker. A
conjugate can
comprise an antibody construct, GS-9620, and a linker. A conjugate can
comprise an antibody
construct, G5K2245035, and a linker. A conjugate can comprise an antibody
construct, TMX-
101, and a linker. A conjugate can comprise an antibody construct, TMX-201,
and a linker. A
conjugate can comprise an antibody construct, TMX-202, and a linker. A
conjugate can comprise
an antibody construct, isatoribine, and a linker. A conjugate can comprise an
antibody construct,
AZD8848, and a linker. A conjugate can comprise an antibody construct,
MEDI9197, and a
linker. A conjugate can comprise an antibody construct, 3M-051, and a linker.
A conjugate can
comprise an antibody construct, 3M-852, and a linker. A conjugate can comprise
an antibody
217
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
construct, 3M-052, and a linker. A conjugate can comprise an antibody
construct, 3M-854A, and
a linker. A conjugate can comprise an antibody construct, S-34240, and a
linker. A conjugate can
comprise an antibody construct, CL663, and a linker. A conjugate can comprise
an antibody
construct, KIN1148, and a linker. A conjugate can comprise an antibody
construct, SB-9200, and
a linker. A conjugate can comprise an antibody construct, KIN-100, and a
linker. A conjugate
can comprise an antibody construct, ADU-S100, and a linker. A conjugate can
comprise an
antibody construct, KU34B, and a linker.
[0497] A conjugate described herein can have a native Fc domain. A conjugate
described herein
can have a modified Fc domain. The modified Fc domain can comprise a
substitution at more
than one amino acid residue such as at 5 different amino acid residues
including
L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including
S239D/I332E, or as at 3 different amino acid residues including
S298A/E333A/K334A. The
numbering of amino acids residues described herein can be according to the EU
index.
[0498] The linker can be a linker as described herein. A linker can be
cleavable, non-cleavable,
hydrophilic, or hydrophobic. A cleavable linker can be sensitive to enzymes. A
cleavable linker
can be cleaved by enzymes such as proteases. A cleavable linker can be a
linker containing a
valine-citrulline or a valine-alanine peptide. A valine-citrulline- or valine-
alanine-containing
linker can contain a pentafluorophenyl group. A valine-citrulline- or valine-
alanine-containing
linker can contain a succimide group. A valine-citrulline- or valine-alanine-
containing linker can
contain a PABA group. A valine-citrulline- or valine-alanine-containing linker
can contain a
PABA group and a pentafluorophenyl group. A valine-citrulline-containing or
valine-alanine-
containing linker can contain a PABA group and a maleimide group. A valine-
citrulline-
containing or valine-alanine-containing linker can contain a PABA group and a
succinimide
group. A non-cleavable linker can be protease insensitive. A non-cleavable
linker can contain a
maleimide group. A non-cleavable linker can be maleimidocaproyl linker. A
maleimidocaproyl
linker can comprise N-maleimidomethylcyclohexane-l-carboxylate. A
maleimidocaproyl linker
can contain a succinimide group. A maleimidocaproyl linker can contain
pentafluorophenyl
group. A linker can be a combination of a maleimide group and one or more
polyethylene glycol
molecules. A linker can be a combination of a maleimidocaproyl group and one
or more
polyethylene glycol molecules. A linker can be a maleimide-PEG4 linker. A
linker can be a
combination of a maleimidocaproyl linker containing a succinimide group and
one or more
polyethylene glycol molecules. A linker can be a combination of a
maleimidocaproyl linker
containing a pentafluorophenyl group and one or more polyethylene glycol
molecules. A linker
can contain maleimides linked to polyethylene glycol molecules in which the
polyethylene glycol
218
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
can allow for more linker flexibility or can be used lengthen the linker. A
linker can be a
(maleimidocaproy1)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker. A
linker can be a
THIOMAB linker. A THIOMAB linker can be a (maleimidocaproy1)-(valine-
citrulline)-(para-
aminobenzyloxycarbonyl) linker. A linker can also comprise an alkylene,
alkenylene, alkynylene,
polyether, polyester, polyamide, polyamino acids, polypeptides, cleavable
peptides, or
aminobenzylcarbamates. A linker can contain a maleimide at one end and an N-
hydroxysuccinimidyl ester at the other end. A linker can contain a lysine with
an N-terminal
amine acetylated, and a valine-citrulline cleavage site. A linker can be a
link created by a
microbial transglutaminase, wherein the link is created between an amine-
containing moiety and
a moiety engineered to contain glutamine as a result of the enzyme catalyzing
a bond formation
between the acyl group of a glutamine side chain and the primary amine of a
lysine chain. A
linker can contain a reactive primary amine. A linker can be a Sortase A
linker. A Sortase A
linker can be created by a Sortase A enzyme fusing an LXPTG recognition motif
(SEQ ID NO:
672) to an N-terminal GGG motif to regenerate a native amide bond. The linker
created can
therefore link a moiety attached to the LXPTG recognition motif (SEQ ID NO:
672) with a
moiety attached to the N-terminal GGG motif. A linker can be a link created
between an
unnatural amino acid on one moiety reacting with oxime bond that was formed by
modifying a
ketone group with an alkoxyamine on another moiety. A moiety can be an
antibody construct. A
moiety can be a binding domain. A moiety can be an antibody. A moiety can be
an immune-
stimulatory compound.
[0499] A conjugate can be an anti-tumor antigen conjugate. The conjugate can
comprise an anti-
tumor antigen antibody or antibody construct. An antigen recognized by the
conjugate can be
CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1,
B7-H3, B7-DC, HLA-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1,
folate-
binding protein, A33, G250, prostate-specific membrane antigen (PSMA),
ferritin, GD2, GD3,
GM2, Leg, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor,
EGRFvIII
(de2-7), EGFR, fibroblast activation protein, tenascin, a metalloproteinase,
endosialin, vascular
endothelial growth factor, avf33, WT1, LMP2, HPV E6, HPV E7, Her-2/neu, p53
nonmutant,
NY-ESO-1, MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl,
tyrosinase,
survivin, PSA, hTERT, a Sarcoma translocation breakpoint fusion protein,
EphA2, PAP, ML-
IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid,
MYCN,
RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, MAGE A3,
sLe(animal),
CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn,
Carbonic anhydrase IX, PAX5, 0Y-TES 1, Sperm protein 17, LCK, HMWMAA, AKAP-4,
219
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
SSX2, XAGE 1, B7H3, Legumain, Tie 3, PAGE4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-
2, ROR2, TRAILl, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, CA6,
NAPI2B, TROP2, Claudin-6 (CLDN6), Claudin-16 (CLDN16), CLDN18.2, RON, LY6E,
FRA,
DLL3, PTK7, Uroplakin-1B (UPK1B), VTCN1 (B7-H4), STRA6, TMPRSS3, TMRRSS4,
TMEM238, Clorf186, LIV1, ROR1, Fos-related antigen 1, VEGFR1, endoglin,
LRRC15,
VISTA, or a fragment thereof. The conjugate can recognize an antigen that can
be expressed on
a cell. The conjugate can recognize an antigen that can be expressed by a
cell. The conjugate can
recognize an antigen that can be expressed in the context of a Major
Histocompatibility
Complex. The conjugate can recognize an antigen that can stimulate activity of
a cell. The
conjugate can recognize an antigen that can stimulate an immune response. The
conjugate can
recognize an antigen that can reduce an immune response. The conjugate can
recognize an
antigen that can reduce activity of a cell. The conjugate can recognize an
antigen that can be
expressed on an immune cell. The conjugate can recognize an antigen that can
be expressed by
an immune cell. The conjugate can recognize an antigen that can be in the
context of a Major
Histocompatibility Complex. The conjugate can recognize an antigen on a cell
wherein the
antigen can be involved in stimulating activity of a cell. The conjugate can
recognize an antigen
on an immune cell that can be involved in the costimulation of an immune cell.
The conjugate
can recognize an antigen on an immune cell that can be involved in the
costimulation of an
immune cell during an immune response. The conjugate can recognize a receptor.
The conjugate
can recognize a receptor on a cell. The conjugate can recognize a receptor
ligand. The conjugate
can recognize a receptor on a cell wherein the receptor can be involved in
stimulating activity of
a cell. The conjugate can recognize a receptor on an immune cell. The
conjugate can recognize a
receptor on an immune cell that can be involved in stimulating activity of an
immune cell. The
conjugate can recognize a receptor on an immune cell that can be involved in
the costimulation
of an immune cell. The conjugate can recognize a receptor on an immune cell
that can be
involved in the costimulation of an immune cell during an immune response. The
conjugate can
recognize an antigen that can be expressed on an immune cell and that can
stimulate activity of
an immune cell. The conjugate can recognize an antigen that can be expressed
on an immune that
can reduce activity of an immune cell. The conjugate can be an anti-CD40
antibody. The
conjugate can comprise a light chain of an SBT-040 antibody. The conjugate can
comprise an
SBT-040-G1WT heavy chain. The conjugate can comprise an SBT-040-G1VLPLL heavy
chain.
The conjugate can comprise an SBT-040-G1DE heavy chain. The conjugate can
comprise an
SBT-040-GlAAA heavy chain. The conjugate can comprise an SBT-040-CDR sequence.
220
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0500] The conjugate can be capable of recognizing a single antigen. The
conjugate can be
capable of recognizing two or more antigens. The conjugate can be capable of
recognizing three
or more antigens. The Kd for binding of a second binding domain of a conjugate
to an antigen in
the presence of an immune-stimulatory compound can be about 2 times, about 3
times, about 4
times, about 5 times, about 6 times, about 7 times, about 8 times, about 9
times, about 10 times,
about 15 times, about 20 times, about 25 times, about 30 times, about 35
times, about 40 times,
about 45 times, about 50 times, about 60 times, about 70 times, about 80
times, about 90 times,
about 100 times, about 110 times, or about 120 times greater than the Kd for
binding of the
second binding domain to the antigen of a conjugate in the absence of the
immune-stimulatory
compound. The Kd for binding of a second binding domain of a conjugate to an
antigen in the
presence of the immune-stimulatory compound can be less than 10 nM. The Kd for
binding of a
second binding domain of a conjugate to an antigen in the presence of the
immune-stimulatory
compound can be less than 100 nM, less than 50 nM, less than 20 nM, less than
5 nM, less than 1
nM, or less than 0.1 nM. In contrast, the Kd for binding of a second binding
domain of conjugate
to an antigen in the presence of the immune-stimulatory compound when the
first binding
domain is bound to the first binding domain's antigen can be greater than 100
nM. The Kd for
binding of a second binding domain of a conjugate to an antigen in the
presence of the immune-
stimulatory compound when the first binding domain is bound to the first
binding domain's
antigen can be greater than 100 nM, greater than 200 nM, greater than 300 nM,
greater than 400
nM, greater than 500 nM, or greater than 1000 nM. The Kd for binding of a
first binding domain
of a conjugate to an antigen in the presence of an immune-stimulatory compound
can be about 2
times, about 3 times, about 4 times, about 5 times, about 6 times, about 7
times, about 8 times,
about 9 times, about 10 times, about 15 times, about 20 times, about 25 times,
about 30 times,
about 35 times, about 40 times, about 45 times, about 50 times, about 60
times, about 70 times,
about 80 times, about 90 times, about 100 times, about 110 times, or about 120
times greater than
the Kd for binding of the first binding domain to the antigen of a conjugate
in the absence of the
immune-stimulatory compound. The Kd for binding of a first binding domain of a
conjugate to
an antigen in the presence of the immune-stimulatory compound can be less than
10 nM. The Kd
for binding of a first binding domain of a conjugate to an antigen in the
presence of the immune-
stimulatory compound can be less than 100 nM, less than 50 nM, less than 20
nM, less than 5
nM, less than 1 nM, or less than 0.1 nM.
[0501] The conjugate can comprise a binding domain. A binding domain of a
conjugate can
recognize an antigen. For example, an antigen can be expressed on an immune
cell. An antigen
can be a peptide or fragment thereof. An antigen can be expressed on an
antigen-presenting cell.
221
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
An antigen can be expressed on a dendritic cell, a macrophage, or a B cell. An
antigen can be
CD40 and a binding domain can recognize a CD40 antigen. A binding domain of a
conjugate can
be a CD40 agonist.
[0502] The conjugate can comprise an Fc domain that can bind to an FcR when
linked to an
immune-stimulatory compound. The conjugate can comprise an Fc domain that can
bind to an
FcR to initiate FcR-mediated signaling when linked to an immune stimulatory
compound. The
conjugate can bind to its antigen when linked to an immune-stimulatory
compound. The
conjugate can bind to its antigen when linked to an immune-stimulatory
compound and the Fc
domain of the conjugate can bind to an FcR when linked to an immune-
stimulatory compound.
The conjugate can bind to its antigen when linked to an immune-stimulatory
compound and the
Fc domain of the conjugate can bind to an FcR to initiate FcR-mediated
signaling when linked to
an immune stimulatory compound. The Fc domain linked to an immune-stimulatory
compound
can be a modified Fc domain. The modified Fc domain can comprise a
substitution at more than
one amino acid residue, such as at 5 different amino acid residues including
L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including
S239D/I332E, or as at 3 different amino acid residues including
S298A/E333A/K334A. The Kd
for binding of an Fc domain to a Fc receptor when the Fc domain is linked to
an immune-
stimulatory compound can be about 2 times, about 3 times, about 4 times, about
5 times, about 6
times, about 7 times, about 8 times, about 9 times, about 10 times, about 15
times, about 20
times, about 25 times, about 30 times, about 35 times, about 40 times, about
45 times, about 50
times, about 60 times, about 70 times, about 80 times, about 90 times, about
100 times, about 110
times, or about 120 times greater than the Kd for binding of the Fc domain to
the Fc receptor in
the absence of linking to the immune-stimulatory compound. The Kd for binding
of an Fc
domain to an Fc receptor when linked to an immune-stimulatory compound can be
less than 10
nM. The Kd for binding of an Fc domain to an Fc receptor when linked to an
immune-
stimulatory compound can be less than 100 nM, less than 50 nM, less than 20
nM, less than 5
nM, less than 1 nM, or less than 0.1 nM. In contrast, the Kd for binding of an
Fc domain to an Fc
receptor when linked to an immune-stimulatory compound and when the first
binding domain is
bound to its antigen can be greater than 100 nM. The Kd for binding of an Fc
domain to an Fc
receptor when linked to an immune-stimulatory compound and when the first
binding domain is
bound to its antigen can be greater than 100 nM, greater than 200 nM, greater
than 300 nM,
greater than 400 nM, greater than 500 nM, or greater than 1000 nM.
[0503] The binding domain can be selected in order to recognize an antigen.
For example, an
antigen can be expressed on an immune cell. An antigen can be expressed on a T
cell, a B cell, an
222
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
NKT cell, or an NK cell. An antigen can be a peptide or fragment thereof. An
antigen can be
expressed on an antigen-presenting cell. An antigen can be expressed on a
dendritic cell, a
macrophage, or a B cell. An antigen can be CD40 and a binding domain can
recognize a CD40
antigen. A binding domain can be a CD40 agonist. A binding domain can be CD40.
[0504] The immune-stimulatory compound of the conjugate can be a PRR agonist.
The PRR
agonist can be a toll-like receptor agonist. The toll-like receptor agonist
can be a TLR1 agonist, a
TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist,
a TLR7
agonist, a TLR8 agonist, a TLR9 agonist, a TLR10 agonist, a TLR11 agonist, a
TLR12 agonist or
a TLR13 agonist. The toll-like receptor agonist can activate two or more TLRs.
The immune-
stimulatory compound of the conjugate can be a PAMP molecule. The PAMP
molecule can be a
RIG-I agonist.
[0505] A conjugate can comprise an antibody construct, KU34B, and a linker. An
antibody
construct of any of the conjugates described herein can have a modified Fc
domain of the
antibody construct. The modified Fc domain can comprise a substitution at more
than one amino
acid residue such as at 5 different amino acid residues including
L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including
S239D/I332E, or as at 3 different amino acid residues including
S298A/E333A/K334A. The
numbering of amino acids residues described herein can be according to the EU
index.
[0506] A conjugate can be formed by a linker that can connect an antibody
construct to an
immune-stimulatory compound. A conjugate can be formed by a linker that can
connect an
antibody construct to a PRR molecule. A conjugate can be formed by a linker
that can connect an
antibody construct to a PAMP molecule. A conjugate can be formed by a linker
that can connect
an antibody construct and a DAMP molecule. A conjugate can be formed by a
linker that can
connect an antibody construct to a PRR, and a linker that can connect an
antibody construct and a
binding domain. A conjugate can be formed by a linker that can connect an
antibody construct to
a PAMP molecule, and a linker that can connect an antibody construct and a
binding domain. A
conjugate can be formed by a linker that can connect an antibody construct and
a DAMP
molecule, and a linker that can connect an antibody construct and a binding
domain.
[0507] A linker can be connected to an antibody construct of a conjugate by a
direct linkage
between the antibody construct and the linker. A linker can be connected to an
anti-CD40
antibody construct by a direct linkage between the anti-CD40 antibody
construct and the linker.
A linker can be connected to an anti-CD40 antibody by a direct linkage between
the anti-CD40
antibody and the linker. A linker can be connected to an anti-tumor antigen
antibody construct by
a direct linkage between the anti-tumor antigen antibody construct and the
linker. A linker can be
223
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
connected to an anti-tumor antigen antibody by a direct linkage between the
anti-tumor antigen
antibody and the linker. A direct linkage is a covalent bond.
[0508] A linker can be attached to an antibody construct at any suitable site,
such as for
example at a terminus of an amino acid sequence or at a side chain of a
cysteine residue, an
engineered cysteine residue, a lysine residue, a serine residue, a threonine
residue, a tyrosine
residue, an aspartic acid residue, a glutamic acid residue, a glutamine
residue, an engineered
glutamine residue, a selenocysteine residue, or a non-natural amino acid. Non-
natural amino
acids can include para-azidomethyl-l-phenylalanine (pAMF). An attachment site
can also be at a
residue containing an oxime bond that was formed by modifying a ketone group
with an
alkoxyamine on another moiety, and a reactive primary amine, such as a
reactive primary amine
at a C-terminal end of a protein or peptide, such as by using Sortase A
linker, which can be
created by a Sortase A enzyme fusing an LXPTG recognition motif (SEQ ID NO:
672) to an N-
terminal GGG motif to regenerate a native amide bond. The linker created can
therefore link a
moiety attached to the LXPTG recognition motif (SEQ ID NO: 672) with a moiety
attached to
the N-terminal GGG motif.
[0509] An attachment can be via any of a number of bonds, for example but not
limited to, an
amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-
carbon single,
double or triple bond, a disulfide bond, or a thioether bond. A linker can
have at least one
functional group, which can be linked to the antibody construct or the
antibody. Non-limiting
examples of the functional groups can include those which form an amide bond,
an ester bond, an
ether bond, a carbonate bond, a carbamate bond, or a thioether bond, such
functional groups can
be, for example, amino groups; carboxyl groups; aldehyde groups; azide groups;
alkyne and
alkene groups; ketones; carbonates; carbonyl functionalities bonded to leaving
groups such as
cyano and succinimidyl and hydroxyl groups.
[0510] A linker can be connected to an antibody construct at a hinge cysteine.
A linker can be
connected to an antibody construct at a light chain constant domain lysine. A
linker can be
connected to an antibody construct at an engineered cysteine in the light
chain. A linker can be
connected to an antibody construct at an engineered light chain glutamine. A
linker can be
connected to an antibody construct at an unnatural amino acid engineered into
the light chain. A
linker can be connected to an antibody construct at a heavy chain constant
domain lysine. A
linker can be connected to an antibody construct at an engineered cysteine in
the heavy chain. A
linker can be connected to an antibody construct at an engineered heavy chain
glutamine. A
linker can be connected to an antibody construct an unnatural amino acid
engineered into the
heavy chain. Amino acids can be engineered into an amino acid sequence of an
antibody
224
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
construct as described herein, for example, and can be connected to a linker
of a conjugate.
Engineered amino acids can be added to a sequence of existing amino acids.
Engineered amino
acids can be substituted for one or more existing amino acids of a sequence of
amino acids.
[0511] A linker can be conjugated to an antibody construct via a sulfhydryl
group. A linker can
be conjugated to an antibody construct via a primary amine. A linker can be a
link created
between an unnatural amino acid on an antibody construct reacting with oxime
bond that was
formed by modifying a ketone group with an alkoxyamine on an immune-
stimulatory compound.
When a linker is connected to an antibody construct at the sites described
herein, an Fc domain of
the conjugate can bind to Fc receptors. When a linker is connected to an
antibody construct at the
sites described herein, the antigen binding domain of the conjugate can bind
its antigen. When a
linker is connected to an antibody construct at the sites described herein, a
binding domain of the
conjugate can bind its antigen.
[0512] An antibody with engineered reactive cysteine residues can be used to
link a binding
domain to the antibody. A linker can connect an antibody construct to a
binding domain via
Sortase A linker. A Sortase A linker can be created by a Sortase A enzyme
fusing an LXPTG
recognition motif (SEQ ID NO: 672) to an N-terminal GGG motif to regenerate a
native amide
bond. The linker created can therefore link an antibody construct attached to
the LXPTG
recognition motif (SEQ ID NO: 672) with a binding domain attached to the N-
terminal GGG
motif. A binding domain can be connected to a linker by a direct linkage. A
direct linkage is a
covalent bond. For example, a linker can be attached to a terminus of an amino
acid sequence of
a binding domain, or could be attached to a side chain modification to the
binding domain, such
as the side chain of a cysteine residue, an engineered cysteine residue, a
lysine residue, a serine
residue, a threonine residue, a tyrosine residue, an aspartic acid residue, a
glutamic acid residue, a
glutamine residue, an engineered glutamine residue, a selenocysteine residue,
or a non-natural
amino acid. Non-natural amino acids can include para-azidomethyl-l-
phenylalanine (pAMF). An
attachment can also be at a residue containing an oxime bond that was formed
by modifying a
ketone group with an alkoxyamine on another moiety, and a reactive primary
amine, such as a
reactive primary amine at a C-terminal end of a protein or peptide. An
attachment can be via any
of a number of bonds, for example but not limited to, an amide bond, an ester
bond, an ether
bond, a carbon-nitrogen bond, a carbon-carbon single double or triple bond, a
disulfide bond, or a
thioether bond. A linker can have at least one functional group, which can be
linked to the
binding domain. Non-limiting examples of the functional groups can include
those which form
an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate
bond, or a thioether
bond, such functional groups can be, for example, amino groups; carboxyl
groups; aldehyde
225
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
groups; azide groups; alkyne and alkene groups; ketones; carbonates; carbonyl
functionalities
bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups.
Amino acids can
be engineered into an amino acid sequence of the binding domain. Engineered
amino acids can
be added to a sequence of existing amino acids. Engineered amino acids can be
substituted for
one or more existing amino acids of a sequence of amino acids. A linker can be
conjugated to a
binding domain via a sulfhydryl group. A linker can be conjugated to a binding
domain via a
primary amine. A binding domain can be conjugated to the C-terminal of an Fc
domain of a
conjugate.
[0513] An antibody or antibody construct with engineered reactive cysteine
residues can be
used to link an immune-stimulatory compound to the antibody or antibody
construct. A linker
can connect an antibody construct to an immune-stimulatory compound via
linker. A linker can
connect an antibody construct to an immune-stimulatory compound via Sortase A
linker. A
Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG
recognition motif (SEQ
ID NO: 672) to an N-terminal GGG motif to regenerate a native amide bond. The
linker created
can therefore link an antibody attached the LXPTG recognition motif (SEQ ID
NO: 672) with an
immune-stimulatory compound attached to the N-terminal GGG motif. A linker can
be a link
created between an unnatural amino acid an antibody reacting with oxime bond
that was formed
by modifying a ketone group with an alkoxyamine on an immune-stimulatory
compound. The
immune-stimulatory compound can comprise one or more rings selected from
carbocyclic and
heterocyclic rings. The immune-stimulatory compound can be covalently bound to
a linker by a
bond to an exocyclic carbon or nitrogen atom on the immune-stimulatory
compound. A linker
can be conjugated to an immune-stimulatory compound via an exocyclic nitrogen
or carbon atom
of an immune-stimulatory compound. A linker can be connected to a STING
agonist, for
example:
,
JII
= Isysks,,,, ===
=====,e
'IrL\ 11
0
HO, P N
P'-=
MM4inlidssAtkilAtet-43 AA GAM
H4 ,{4
J O1
\op
0 3
y- 4
0:
226
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
9, 0 0
vm = =-= 0 '... ' 0 '", il ..sse 4
mt4. ,,,õ=-= 'so ., =,..se.v. .....,...µ \ eod," ,e, \ .õ., .= ,,,,,,, 1N e
,....,,,eN =seN,, ,...,,,
i H I, il
Ns.,..,-'=\)N = ¨;>=-
rk
HO,
OH 0--..1
WMtrth:.te.PEC:4GAMP
Hzisl,v.vt4 0
11 \...v.* ,.....,
i s>
i N
0 ,
0
NI A.
-4
-"--
e> ii NH :
\ .,..,k .K.1.
HO. 9 N 'N' -NH2
P-0 .,. 0 0
9H
H i H $s
1
õõ..,=:,. Ns
-,'"*::::"*"µ ' = :.
..,"
., ,..:. 0
=\,...:,
i o¨p.../..
Hz.N..,
lsis.
1r hi
0 w"ieimgdo-Vat.Akk-PASA-c-G(r-Mt4P-GP
,
0
13\
.3'1"Yr. 'NH
< i I
HO.... .. P.,
'1 N NH2
õP ¨0,
, =..- ) 1 i
QH 0- 0
/; ".,..
i '"'0' r" 0
,....(),,, ,, ,..,-,..0 s.--'\., eN'N ....`''''''Ness.."0/
õ if õ ,.. 0 õ ,.. , .. 4
:. ¨P.
Wsi.,,es.N. e \ Azi $o14 0 6 0
HA.. ,....!4.,4'
II '
0 Wdeitnkie-PE,,G4-4(2'4,411,z)p-GP .
[0514] A linker agonist complex can dissociate under physiological conditions
to yield an active
agonist.
[0515] A linker can be connected to a PRR agonist by a direct linkage between
the PRR agonist
and the linker. A linker can be connected to a PAMP molecule by a direct
linkage between the
PAMP molecule and the linker. A linker can be connected to a toll-like
receptor agonist by a
direct linkage between the toll-like receptor agonist and the linker.
[0516] Examples of toll-like receptor agonists connected to a linker in a
manner able to release
an active toll-like receptor agonist under physiologic conditions can include:
227
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0
& jnNIN.,cinrH
N 0 0 crH 0 00 AN
H
N NC1\1 N H
0 I '.)1\1
N \ H
I ,N
0 0
NH2
0
NH2
Maleimide-PEG4-KU34b Maleimide-Val-Ala-PABA-
KU34b
0
0 0
H
cf'0)0-1'N-CHr N 0 0 0 ill AN
0 4
0 IN H ii H
N,"N N
NH2 H H I x N
0 0
0
NHS-PEG5-KU34b NHS-Val-Ala-PABA-KU34b
NH2
[0517] Examples of RIG-I agonists connected to a linker in a manner able to
release an active
toll-like receptor agonist under physiologic conditions can include:
0
s
"==== FI\1'rrll?
H 4 0 0
lir 0 'N
ill
OCH3
Maleimide-PEG4-KIN700
0
CI
S Nyt.............,0, 0(0,1-
H 5 0
0 '`N
ill
OCH3
NHS-PEGS-KIN700
S 0
41
CI NAO 0 h H 0 0
\
0 '''' I \ I H
H
Si CH3 0 0
OCH3
Maleimide-Val-Ala-PABC-KIN700
S 0
CI
SI N AO
H 0
0 '`N ISI N1)YMI).r 'A
H 11
00 CH3 0 0
C:r
OCH3
NHS-Val-Ala-PABC-KIN700
228
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0518] A linker can be connected to a DAMP molecule by a direct linkage
between the DAMP
molecule and the linker. A direct linkage can be a covalent bond. For example,
a linker can be
attached to a terminus of an amino acid sequence of an antibody or antibody
construct, or could
be attached to a side chain modification to the antibody or antibody
construct, such as example at
a side chain of a cysteine residue, an engineered cysteine residue, a lysine
residue, a serine
residue, a threonine residue, a tyrosine residue, an aspartic acid residue a
glutamic acid residue, a
glutamine residue, an engineered glutamine residue, a selenocysteine residue,
or a non-natural
amino acid. Non-natural amino acids can include para-azidomethyl-l-
phenylalanine (pAMF). An
attachment can also be at a residue containing an oxime bond that was formed
by modifying a
ketone group with an alkoxyamine on another moiety, and a reactive primary
amine, such as a
reactive primary amine at a C-terminal end of a protein or peptide, such as by
using Sortase A
linker, which can be created by a Sortase A enzyme fusing an LXPTG recognition
motif (SEQ
ID NO: 672) to an N-terminal GGG motif to regenerate a native amide bond. The
linker created
can therefore link a moiety attached to the LXPTG recognition motif (SEQ ID
NO: 672) with a
moiety attached to the N-terminal GGG motif. An attachment can be via any of a
number of
bonds, for example but not limited to, an amide bond, an ester bond, an ether
bond, a carbon-
nitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond,
or a thioether
bond. A linker can have at least one functional group, which can be linked to
the antibody
construct. Non-limiting examples of the functional groups can include those
which form an
amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond,
or a thioether
bond, such functional groups can be, for example, amino groups; carboxyl
groups; aldehyde
groups; azide groups; alkyne and alkene groups; ketones; carbonates; carbonyl
functionalities
bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups.
[0519] In some embodiments, the linker is not attached to an amino acid
residue of the Fc
domain of the antibody construct selected from a group consisting of: 221,
222, 224, 227, 228,
230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245,
246, 247, 249, 250,
258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,
276, 278, 280, 281,
283, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,
302, 305, 313, 317,
318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335
336, 396, or 428,
wherein numbering of amino acid residues in said Fc domain is according to the
EU index as in
Kabat. In some embodiments, the linker is not attached to an amino acid
residue of the Fc
domain of the antibody construct selected from a group consisting of: 221,
224, 227, 230, 231,
232, 234, 235, 236, 237, 239, 240, 243, 244, 245, 247, 249, 258, 262, 263,
264, 265, 266, 267,
268, 269, 270, 271, 272, 273, 275, 278, 280, 281, 283, 285, 286, 291, 292,
293, 294, 295, 296,
229
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
297, 298, 299, 300, 305, 313, 323, 324, 325, 327, 328, 329, 330, 331, 332,
333, 335, 336, 396, or
428, wherein numbering of amino acid residues in said Fc domain is according
to the EU index
as in Kabat. In some embodimentsõ wherein the linker is covalently bound to a
residue of the
antibody construct selected from the group consisting of a lysine residue,
cysteine residue, and a
glutamine residue, or is covalently bound to said antibody construct using a
Sortase A linker.
[0520] In some embodiments, a linker-immune-stimulatory compound (an ATAC) can
be
formed by conjugating a noncleavable maleimide-PEG4 linker containing a
succinimide group
with an immune-stimulatory compound. For example, an ATAC can be N-((4-amino-1-
(2-
hydroxy-2-methylpropy1)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)-1-(3-(2,5-dioxo-
2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide
(ATAC11); N-(5-
(2-amino-3-pentylquinolin-5-yl)penty1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC12); 1-(3-(2,5-dioxo-
2,5-
dihydro-1H-pyrrol-1-yl)propanamido)-N-(3-pentylquinolin-2-y1)-3,6,9,12-
tetraoxapentadecan-
15-amide (ATAC13); 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
(1-isobutyl-
1H-imidazo[4,5-c]quinolin-4-y1)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC14);
1-(3-(2,5-
dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-methyl-N-(2-(3-(7-
methylbenzo[1,2-d:3,4-
0bis(thiazole)-2-yOureido)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide
(ATAC15); (S)-1-(3-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(1-((7-methylbenzo[1,2-
d:3,4-
dlbis(thiazole)-2-yflamino)-1-oxo-3-phenylpropan-2-y1)-3,6,9,12-
tetraoxapentadecan-15-amide
(ATAC16); N-(benzo[d]thiazol-2-y1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
y1)propanamido)-
N-((8-hydroxyquinolin-7-y1)(4-(trifluoromethoxy)phenyl)methyl)-3,6,9,12-
tetraoxapentadecan-
15-amide (ATAC17); N-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9-bis(2-amino-6-oxo-
1H-purin-
9(6H)-y1)-5,10,12-trihydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-
j][1,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-3-y1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-
3,6,9,12-tetraoxapentadecan-15-amide (ATAC18); N-
((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9-
bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-10-hydroxy-5,12-dimercapto-5,12-
dioxidodecahydrodifuro[3,2-d:3',2'-
j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-3-y1)-1-(3-(2,5-
dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-
amide
(ATAC19); N-(9-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-9-(2-amino-6-oxo-1H-purin-
9(6H)-y1)-
3,5,10,12-tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2*-
A[1,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC20); or N-(9-
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo-1H-purin-9(6H)-y1)-3
,5,10,12-
tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-
230
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-
dioxo-2,5-dihydro-
1H-pyrrol-1-y1)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC21).
[0521] An ATAC can be formed by conjugating a cleavable linker containing a
valine-alanine
or valine-citrulline dipeptide, a PABA group and a maleimide group with an
immune-stimulatory
compound. For example, an ATAC can be 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-
dihydro-1H-pyrrol-
1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl ((4-amino-1-(2-hydroxy-
2-methyl-
propy1)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate (ATAC22); 4-
((S)-2-((S)-2-(6-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methyl-
butanamido)propanamido)benzyl
(5-(2-amino-3-pentylquinolin-5-yl)penty1)-carbamate (ATAC23); 4-((S)-2-((S)-2-
(6-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutan-amido)-5-
ureidopentanamido)benzyl-
(5-(2-amino-3-pentylquinolin-5-yl)penty1)-carbamate (ATAC24); 4-((S)-2-((S)-2-
(6-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-
ureidopentanamido)benzyl((4-
amino-1-(2-hydroxy-2-methylpropy1)- 1H-imidazo[4,5-c]quinolin-2-
yl)methyl)(ethyl)carbamate
TFA salt (ATAC25); 2-(3-124N-Methyle[p-RS)-2-1(S)-246-(2,5-dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-methylbutyrylamino}-5-
ureidovalerylamino]phenyl} metho xycarbonyl)amino] ethyl } ureido)-7-methyl-
1,6-dithia-3 ,8-
diaza-as-indacene (ATAC26); 2-1 [(8-Hydroxy-7-quinoly1)(p-
trifluoromethoxyphenyOmethyl] (1 p- RS )-2-1 (S)-2-[6-(2,5-dioxo-1H-pyrrol-1-
yOhexanoylamino]-
3-methylbutyrylamino }-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino } -
1,3-
benzothiazole (ATAC27); (1R,6R,8R,95,10S,15R,17R,18S)-18-(1p-RS)-2-1(S)-246-
(2,5-Dioxo-
1H-pyrrol-1-y1)hexanoylamino]-3-methylbutyrylamino}-5-
ureidovalerylamino]phenyl} methoxycarbonylamino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-
9-y1)-3,12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione (ATAC28);
(1R,6R,8R,95,10S,15R,17R,18S)-18-(1p-RS)-2-1(S)-246-(2,5-Dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-methylbutyrylamino
}propionylamino]phenyl}methoxycarbonylamino)-
8,17-bis(2-amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-9-hydroxy-
2.4.7.11.13.16-
hexaoxa-3k5.1215-diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione
(ATAC29);
(1R,6R,8R,95,10S,15R,17R,18S)-18-(1p-RS)-2-1(S)-246-(2,5-Dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-methylbutyrylamino}-5-
ureidovalerylamino]phenyl} methoxycarbonylamino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-
9-y1)-9-hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione (ATAC30); 1p- [(S)-2-1
(S)-246-(2,5-
Dioxo-1H-pyrrol-1-yOhexanoylamino]-3-methylbutyrylamino }-5-
231
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
ureidovalerylamino]phenyl} methyl 9-1 (1S ,6R,8R,9S ,10S ,15R,17R,18S )-8-(2-
amino-6-oxo- 1,9-
dihydropurin-9-y1)-3,12-dihydroxy-9,18-dihydroxy-3,12-dioxo-2.4.7.11.13.16-
hexaoxa-
3k5 .1215-dipho sphatric yclo [13 .2.1.06,10]octadec-17- yl} -9a-adenineecarbo
xylate (ATAC31; 1-
I 6- R I 7-Amino-3-(2-hydroxy-2-methylpropy1)-3.5.8-triazatricyclo [7
.4Ø02,6]tridec a-
1(9),2(6),4,7,10,12-hexaen-4- yl} methyl)-N-ethylamino] -6-o xohexyl } -1H-
pyrrole-2,5-dione
(ATAC32); 1- I [441 6- R I 7-Amino-3-(2-hydroxy-2-methylpropy1)-3 .5.8-
triazatricyclo [7 .4Ø02,6]trideca-1(9),2(6),4,7,10,12-hexaen-4-y1} methyl)-N-
ethylamino] -6-
oxohexylamino }carbonyl)cyclohexyl]methy1}-1H-pyrrole-2,5-dione (ATAC33); or
14(4- I RI 7-
Amino-3-(2-hydroxy-2-methylpropy1)-3.5.8-triazatricyclo[7.4Ø02,6]trideca-
1(9),2(6),4,7,10,12-
hexaen-4- yl} methyl)-N-ethylamino] -carbonyl } c yclohexyl)methyl] - 1H-p
yrro le-2,5-dione
(ATAC34).
[0522] An ATAC can be formed by conjugating a noncleavable maleimide-PEG4
linker
containing an activated ester such as a pentafluorophenyl group or an N-
hydroxysuccinimide
group with an immune-stimulatory compound. For example, an ATAC can be
pentafluorophenyl
25-(2-amino-3-pentylquinolin-5-y1)-19-oxo-4,7,10,13,16-pentaoxa-20-
azapentacosanoate
(ATAC1); perfluorophenyl 3-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-
imidazo[4,5-
c]quinolin-2-y1)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oate
(ATAC2);
pentafluorophenyl 25-(2-amino-3-pentylquinolin-5-y1)-19-oxo-4,7,10,13,16-
pentaoxa-20-
azapentacosanoate (ATAC3); or 2,5-Dioxopyrrolidin-1-y134(4-amino-1-(2-hydroxy-
2-
methylpropy1)-1H-imidazo-[4,5-c]quinolin-2-y1)methyl)-4-oxo-7,10,13,16,19-
pentaoxa-3-
azadocosan-22-oate (ATAC4).
[0523] An ATAC can be formed by conjugating a cleavable linker containing a
valine-alanine
or valine-citrulline dipeptide, a PABA group and an activated ester such as a
pentafluorophenyl
group or an N-hydroxysuccinimde group to an immune-stimulatory compound. For
example, an
ATAC can be 2,5-dioxopyrrolidin-1-y1 6-(((S)-1-(((S)-1-((4-((((5-(2-amino-3-
pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-
1-oxobutan-
2-yl)amino)-6-oxohexanoate (ATAC5); 2,5-dioxopyrrolidin-l-y17-(((S)-1-(((S)-1-
((4-(((((4-
amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo [4,5-c] quinolin-2-
yl)methyl)(ethyl)c arbamo yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-
3-methy1-1-
oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC6); 2,5-dioxopyrrolidin-l-y17-(((S)-
1-(((S)-1-((4-
(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo [4,5-c] quino lin-2-
yl)methyl)(ethyl)c arbamo yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC7); perfluorophenyl 6-(((S)-
1-(((S)-1-
((4-((((5-(2-amino-3-pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-
232
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
oxopropan-2-yl)amino)-3-methy1-1-oxobutan-2-y1)amino)-6-oxohexanoate (ATAC8);
perfluorophenyl 7-(((S)-1-(((S)-1-((4-(((((4-amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-
2-yl)amino)-3-methyl-1-oxobutan-2-y1)amino)-7-oxoheptanoate (ATAC9); or
perfluorophenyl 7-
(((S)-1-(((S)-1-((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-
c]quinolin-2-
yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC10).
[0524] An antibody construct of a conjugate can comprise an anti-CD40
antibody. An anti-
CD40 antibody can comprise two SBT-040-G1WT heavy chains and two light chains
from a
SBT-040 antibody, which can be referred to as SBT-040-WT. An anti-CD40
antibody can
comprise two SBT-040-G1VLPLL heavy chains and two light chains from a SBT-040
antibody,
which can be referred to as SBT-040-VLPLL. An anti-CD40 antibody can comprise
two SBT-
040-G1DE heavy chains and two light chains from a SBT-040 antibody, which can
be referred to
as SBT-040-DE. An anti-CD40 antibody can comprise two SBT-040-G1AAA heavy
chains and
two light chains from a SBT-040 antibody, which can be referred to as SBT-040-
AAA. An anti-
CD40 antibody can comprise two IgG2 heavy chains and two light chains from a
SBT-040
antibody, which can be referred to as SBT-040-G2.
[0525] A conjugate can comprise SBT-040-WT-ATAC1. A conjugate can comprise SBT-
040-
WT-ATAC2. A conjugate can comprise SBT-040-WT-ATAC3. A conjugate can comprise
SBT-
040-WT-ATAC4. A conjugate can comprise SBT-040-WT-ATAC5. A conjugate can
comprise
SBT-040-WT-ATAC6. A conjugate can comprise SBT-040-WT-ATAC7. A conjugate can
comprise SBT-040-WT-ATAC8. A conjugate can comprise SBT-040-WT-ATAC9. A
conjugate
can comprise SBT-040-WT-ATAC10. A conjugate can comprise SBT-040-WT-ATAC11. A
conjugate can comprise SBT-040-WT-ATAC12. A conjugate can comprise SBT-040-WT-
ATAC13. A conjugate can comprise SBT-040-WT-ATAC14. A conjugate can comprise
SBT-
040-WT-ATAC15. A conjugate can comprise SBT-040-WT-ATAC16. A conjugate can
comprise SBT-040-WT-ATAC17. A conjugate can comprise SBT-040-WT-ATAC18. A
conjugate can comprise SBT-040-WT-ATAC19. A conjugate can comprise SBT-040-WT-
ATAC20. A conjugate can comprise SBT-040-WT-ATAC21. A conjugate can comprise
SBT-
040-WT-ATAC22. A conjugate can comprise SBT-040-WT-ATAC23. A conjugate can
comprise SBT-040-WT-ATAC24. A conjugate can comprise SBT-040-WT-ATAC25. A
conjugate can comprise SBT-040-WT-ATAC26. A conjugate can comprise SBT-040-WT-
ATAC27. A conjugate can comprise SBT-040-WT-ATAC28. A conjugate can comprise
SBT-
040-WT-ATAC29. A conjugate can comprise SBT-040-WT-ATAC30. A conjugate can
233
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
comprise SBT-040-WT-ATAC31. A conjugate can comprise SBT-040-WT-ATAC32. A
conjugate can comprise SBT-040-WT-ATAC33. A conjugate can comprise SBT-040-WT-
ATAC34. A conjugate can comprise SBT-040-VLPLL-ATAC1. A conjugate can comprise
SBT-
040-VLPLL-ATAC2. A conjugate can comprise SBT-040-VLPLL-ATAC3. A conjugate can
comprise SBT-040-VLPLL-ATAC4. A conjugate can comprise SBT-040-VLPLL-ATAC5. A
conjugate can comprise SBT-040-VLPLL-ATAC6. A conjugate can comprise SBT-040-
VLPLL-
ATAC7. A conjugate can comprise SBT-040-VLPLL-ATAC8. A conjugate can comprise
SBT-
040-VLPLL-ATAC9. A conjugate can comprise SBT-040-VLPLL-ATAC10. A conjugate
can
comprise SBT-040-VLPLL-ATAC11. A conjugate can comprise SBT-040-VLPLL-ATAC12.
A
conjugate can comprise SBT-040-VLPLL-ATAC13. A conjugate can comprise SBT-040-
VLPLL-ATAC14. A conjugate can comprise SBT-040-VLPLL-ATAC15. A conjugate can
comprise SBT-040-VLPLL-ATAC16. A conjugate can comprise SBT-040-VLPLL-ATAC17.
A
conjugate can comprise SBT-040-VLPLL-ATAC18. A conjugate can comprise SBT-040-
VLPLL-ATAC19. A conjugate can comprise SBT-040-VLPLL-ATAC20. A conjugate can
comprise SBT-040-VLPLL-ATAC21. A conjugate can comprise SBT-040-VLPLL-ATAC22.
A
conjugate can comprise SBT-040-VLPLL-ATAC23. A conjugate can comprise SBT-040-
VLPLL-ATAC24. A conjugate can comprise SBT-040-VLPLL-ATAC25. A conjugate can
comprise SBT-040-VLPLL-ATAC26. A conjugate can comprise SBT-040-VLPLL-ATAC27.
A
conjugate can comprise SBT-040-VLPLL-ATAC28. A conjugate can comprise SBT-040-
VLPLL-ATAC29. A conjugate can comprise SBT-040-VLPLL-ATAC30. A conjugate can
comprise SBT-040-VLPLL-ATAC31. A conjugate can comprise SBT-040-VLPLL-ATAC32.
A
conjugate can comprise SBT-040-VLPLL-ATAC33. A conjugate can comprise SBT-040-
VLPLL-ATAC34. A conjugate can comprise SBT-040-VLPLL-ATAC34. A conjugate can
comprise SBT-040-DE-ATAC1. A conjugate can comprise SBT-040-DE-ATAC2. A
conjugate
can comprise SBT-040-DE-ATAC3. A conjugate can comprise SBT-040-DE-ATAC4. A
conjugate can comprise SBT-040-DE-ATAC5. A conjugate can comprise SBT-040-DE-
ATAC6.
A conjugate can comprise SBT-040-DE-ATAC7. A conjugate can comprise SBT-040-DE-
ATAC8. A conjugate can comprise SBT-040-DE-ATAC9. A conjugate can comprise SBT-
040-
DE-ATAC10. A conjugate can comprise SBT-040-DE-ATAC11. A conjugate can
comprise
SBT-040-DE-ATAC12. A conjugate can comprise SBT-040-DE-ATAC13. A conjugate can
comprise SBT-040-DE-ATAC14. A conjugate can comprise SBT-040-DE-ATAC15. A
conjugate can comprise SBT-040-DE-ATAC16. A conjugate can comprise SBT-040-DE-
ATAC17. A conjugate can comprise SBT-040-DE-ATAC18. A conjugate can comprise
SBT-
040-DE-ATAC19. A conjugate can comprise SBT-040-DE-ATAC20. A conjugate can
comprise
234
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
SBT-040-DE-ATAC21. A conjugate can comprise SBT-040-DE-ATAC22. A conjugate can
comprise SBT-040-DE-ATAC23. A conjugate can comprise SBT-040-DE-ATAC24. A
conjugate can comprise SBT-040-DE-ATAC25. A conjugate can comprise SBT-040-DE-
ATAC26. A conjugate can comprise SBT-040-DE-ATAC27. A conjugate can comprise
SBT-
040-DE-ATAC28. A conjugate can comprise SBT-040-DE-ATAC29. A conjugate can
comprise
SBT-040-DE-ATAC30. A conjugate can comprise SBT-040-DE-ATAC31. A conjugate can
comprise SBT-040-DE-ATAC32. A conjugate can comprise SBT-040-DE-ATAC33. A
conjugate can comprise SBT-040-DE-ATAC34. A conjugate can comprise SBT-040-AAA-
ATAC1. A conjugate can comprise SBT-040-AAA-ATAC2. A conjugate can comprise
SBT-
040-AAA-ATAC3. A conjugate can comprise SBT-040-AAA-ATAC4. A conjugate can
comprise SBT-040-AAA-ATAC5. A conjugate can comprise SBT-040-AAA-ATAC6. A
conjugate can comprise SBT-040-AAA-ATAC7. A conjugate can comprise SBT-040-AAA-
ATAC8. A conjugate can comprise SBT-040-AAA-ATAC9. A conjugate can comprise
SBT-
040-AAA-ATAC10. A conjugate can comprise SBT-040-AAA-ATAC11. A conjugate can
comprise SBT-040-AAA-ATAC12. A conjugate can comprise SBT-040-AAA-ATAC13. A
conjugate can comprise SBT-040- AAA -ATAC14. A conjugate can comprise SBT-040-
AAA-
ATAC15. A conjugate can comprise SBT-040-AAA-ATAC16. A conjugate can comprise
SBT-
040-AAA-ATAC17. A conjugate can comprise SBT-040-AAA-ATAC18. A conjugate can
comprise SBT-040-AAA-ATAC19. A conjugate can comprise SBT-040-AAA-ATAC20. A
conjugate can comprise SBT-040-AAA-ATAC21. A conjugate can comprise SBT-040-
AAA-
ATAC22. A conjugate can comprise SBT-040-AAA-ATAC23. A conjugate can comprise
SBT-
040-AAA-ATAC24. A conjugate can comprise SBT-040-AAA-ATAC25. A conjugate can
comprise SBT-040-AAA-ATAC26. A conjugate can comprise SBT-040-AAA-ATAC27. A
conjugate can comprise SBT-040-AAA-ATAC28. A conjugate can comprise SBT-040-
AAA-
ATAC29. A conjugate can comprise SBT-040-AAA-ATAC30. A conjugate can comprise
SBT-
040-AAA-ATAC31. A conjugate can comprise SBT-040-AAA-ATAC32. A conjugate can
comprise SBT-040-AAA-ATAC33. A conjugate can comprise SBT-040-AAA-ATAC34. The
Kd
for binding of the CD40 binding domain of any of these conjugates to CD40 can
be about 2
times, about 3 times, about 4 times, about 5 times, about 6 times, about 7
times, about 8 times,
about 9 times, about 10 times, about 15 times, about 20 times, about 25 times,
about 30 times,
about 35 times, about 40 times, about 45 times, about 50 times, about 60
times, about 70 times,
about 80 times, about 90 times, about 100 times, about 110 times, or about 120
times greater than
the Kd for binding of the CD40 binding domain to CD40 in the absence of the
immune-
stimulatory compound or ATAC. The Kd for binding of the CD40 binding domain of
any of
235
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
these conjugates to CD40 can be less than 10 nM. The Kd for binding of the
CD40 binding
domain of any of the conjugates to CD40 can be less than 100 nM, less than 50
nM, less than 20
nM, less than 5 nM, less than 1 nM, or less than 0.1 nM. The Kd for binding of
the Fc domain of
any of the conjugates to an Fc receptor can be about 2 times, about 3 times,
about 4 times, about
times, about 6 times, about 7 times, about 8 times, about 9 times, about 10
times, about 15
times, about 20 times, about 25 times, about 30 times, about 35 times, about
40 times, about 45
times, about 50 times, about 60 times, about 70 times, about 80 times, about
90 times, about 100
times, about 110 times, or about 120 times greater than the Kd for binding of
the Fc domain to
the Fc receptor in the absence of the immune-stimulatory compound or ATAC. The
Kd for
binding of the Fc domain of any of the conjugates to an Fc receptor can be
less than 10 nM. The
Kd for binding of the Fc domain of any of the conjugates to an Fc receptor can
be less than 100
nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less
than 0.1 nM.
[0526] In a conjugate, an antibody can be linked to an immune-stimulatory
compound in such a
way that the antibody can still bind to an antigen and the Fc domain of the
antibody can still bind
to an FcR or FcR-mediated signaling resulting from the Fc domain of the
antibody from binding
to an FcR. In a conjugate, an antibody construct is linked to an immune-
stimulatory compound in
such a way that the linking does not interfere with ability of the antigen
binding domain of the
antibody construct to bind to antigen, the ability of the Fc domain of the
antibody construct to
bind to an FcR, or FcR-mediated signaling resulting from the Fc domain of the
antibody
construct from binding to an FcR. In a conjugate, an immune-stimulatory
compound can be
linked to an antibody construct in such a way the linking does not interfere
with the ability of the
immune-stimulatory compound to bind to its receptor. A conjugate can produce
stronger immune
stimulation and a greater therapeutic window than components of the conjugate
alone. In an anti-
CD40 antibody linked to a TLR agonist conjugate, the combination of CD40
agonism, TLR
agonism, and an accessible Fc domain of the anti-CD40 antibody to allow FcR-
mediated
signaling can produce stronger immune stimulation and a greater therapeutic
window than the
CD40 agonism, TLR agonism, or the FcR-mediated signaling alone.
[0527] In some embodiments, the recombinant bispecific antibody further
comprises an immune
stimulatory compound and a linker, wherein the linker links the immune-
stimulatory compound
to the Fc comprising domain.
[0528] In some aspects, a method of making a conjugate comprises linking an
antibody construct
as described herein to an immune stimulatory compound by a linker.
Synthesis of Immune-Stimulatory Compounds
236
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0529] An immune stimulatory compound can be synthesized as shown in Scheme
Al.
Scheme Al:
O 1. Tf,o o o
N-----AN-H 2. NaNO2 / H20 N----.),N-H 1. DMTrCI
N----)LN-H
1 ,I, 3. Tf20
_______________________________ ' 1
,, 2. H2 1
,,
N----'N--. NH'Bu ,,---'N NH'Bu ______ . ,,---'N
NH'Bu
4. NaN3
4 3. CbzCI
j...
A NHCbz
' OH 5. TBAF ' ' ,N3 4. OEM
DMTrO
O b HO bH 0-p'a
ssi,cys,i /
/-----./ 1)¨
NON
-1 \
i iii
1. TFA/pyr
o 2. deprotect
0
N-----AN-H
N ----)I,N - H 3.
deprotect
1 oNAN"
N N NH'Bu ,,
.---'N NH'Bu NI N'LNH'Bu o
OH NHCbz ON DmTro...4....,OTBS
N-._.)t..N,H
1. oxidize
-.....NHCbz ,....,o prSi-' 1
0.-.P\ b\ pH 2. deprotect L ..,..0
P NC ...\ -(
N----'N NH'Bu
"--Q 3. cyclize \--. bP02H
j...
o =0
NHCbz
TBSO''' cr: ODMTr HO
TBSO'''
oP02H
iBuHN,N N
I 1 'BuHN N N
H-Ny---N VI
H-Ny---N V IV
o
o
1. hydrogenate
2. deprotect
o o
N-.....--ILN-H
N N NH2 N----'N NH2
. j... j,..
OH " 'NH2 OH " 'NH2
0-'''P\ deprotect a\ pH 0"-'"--Q P\ a\ pH
w¨c2
'c-ro =0 ' 'c--0
TBSO''' HO'',
0
H2N,4...N...,__N H2N N...,__N
I 1 vii 1 H-Ny"--N H-N-r-N VIII
0 0
[0530] Synthesis of the C-2' amino cyclic dinucleotide (viii) can be
accomplished using a
multistep synthesis as outlined in scheme Al above.
Synthesis of Exemplary ATAC Compounds
[0531] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC1 ¨ ATAC4, can be synthesized as shown in Scheme Bl.
Scheme Bl:
237
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
1.
0 0
H2N
HO X R 41,
n0
2. DC/ROH
R = NHS, pentafluorophenyl
ISO: immune-stimulatory compound
[0532] A PEGylated carboxylic acid (i) that has been activated for amide bond
formation can be
reacted with an appropriately substituted amine containing immune-stimulatory
compound to
afford an intermediate amide. Formation of an activated ester (ii) can be
achieved by reaction the
intermediate amide-containing carboxylic using a reagent such as N-
hydroxysuccinimide or
pentafluorophenol in the presence of a coupling agent such as
diisopropylcarbodiimide (DIC) to
provide compounds (ii).
[0533] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC5 ¨ ATAC10, can be synthesized as shown in Scheme B2.
Scheme B2:
010 411)
0 R1 Hi H2N OI
i N
40 0 Ri H 0
H
0 0 R2
0 R2
NO2
deprotect
4111 0 NI DAN H
0 R1
couple CA
R40 N 411) H
0
___________________________________________ Halr,1õHjyNyliõN H
0 0 R2
0 0 R2
IN/
R4 = NHS, Perfluorofenyl
ISC: immune-stimulatory compound
[0534] An activated carbonate such as (i) can be reacted with an appropriately
substituted amine
containing immune-stimulatory compound to afford carbamates (ii) which can be
deprotected
using standard methods based on the nature of the R3 ester group. The
resulting carboxylic acid
(iii) can then by coupled with an activating agent such as N-
hydroxysuccinimide or
pentafluorophenol to provide compounds (iv).
[0535] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC11 ¨ ATAC21, can be synthesized as shown in Scheme B3.
238
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Scheme B3:
0 0
0 0
H2N
0
0 0 0 0
i-a; X = NHS
i-b; X = H
ISC: immune-stimulatory compound
[0536] An activated carboxylic ester such as (i-a) can be reacted with an
appropriately
substituted amine containing immune-stimulatory compound to afford amides
(ii). Alternatively,
carboxylic acids of type (i-b) can be coupled to an appropriately substituted
amine containing
immune-stimulatory compound in the presence of an amide bond forming agent
such as
dicyclohexycarbodiimde (DCC) to provide the desired ATAC compounds.
[0537] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC22 ¨ ATAC31, can be synthesized as shown in Scheme B4.
Scheme B4:
cro o 110 o o H2N
40
0 0 R2
NO2
A
0
N 111 N * 0 N
Y ¨H
0 0 R2
ISO: immune-stimulatory compound
[0538] An activated carbonate such as (i) can be reacted with an appropriately
substituted amine
containing immune-stimulatory compound to afford carbamates (ii) as the target
ATAC
compounds.
[0539] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC32 ¨ ATAC34, can be synthesized as shown in Scheme B5.
Scheme B5:
239
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
H2N
JLN
0
0
i-a ii-a
0
0
H2 N
0
0
0
0
i-b ii-b
0 H2 CD
cue N x _______________ 0 ilk
yip
0 0
i-c ii-c
ISC: immune-stimulatory compound
[0540] An activated carboxylic acid such as (i-a, i-b, i-c) can be reacted
with an appropriately
substituted amine containing immune-stimulatory compound to afford amides (ii-
a, ii-b, ii-c) as
the target ATAC compounds.
Synthesis of Exemplary TLR8 Agonist Compounds
[0541] The following synthetic schemes are provided for purposes of
illustration, not limitation.
The following examples illustrate the various methods of making compounds
described herein.
It is understood that one skilled in the art may be able to make these
compounds by similar
methods or by combining other methods known to one skilled in the art. It is
also understood that
one skilled in the art would be able to make, in a similar manner as described
below by using the
appropriate starting materials and modifying the synthetic route as needed. In
general, starting
materials and reagents can be obtained from commercial vendors or synthesized
according to
sources known to those skilled in the art or prepared as described herein.
240
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Scheme Cl: Synthesis of C-8 Carboxamide
o o o
reductive NH2
deprotect
is H3C0 NO2 condensation H 3co NO2 N._
_,....
_,.. cyclization
H3C0
NC I CO2tBu
CO2tBu
i ii ill
0 0 0
NH2 NH2 NHBoc
N._ couple N._ protect N._
hydrolyze
H3C0 -A- H3C0 -).- H3C0 _,,..
-- -- --
CO2H NRR' NRR'
0 0
iV V Vi
0 0 0
NHBoc NH2
NHBoc
N._
HO couple RR"N deprotect RR"N
_),.... _,..
-- --
--
NRR' NRR' NRR'
0 0 0
vii Vill ix
[0542] React an aldehyde (i) with an appropriately Wittig reagent, such as
tert-butyl 3-cyano-2-
(triphenylphosphorylidene)propanoate, at elevated temperatures to afford an
olefin (ii), which
undergoes reductive cyclization by treating the olefin (ii) with a reducing
agent, such as iron
powder in hot acetic acid, to afford azepines (iii). Deprotect the C-4 ester
group by using a
strong acid such as HC1 to give compounds (iv), which is in turn coupled with
a substituted
amine using a coupling agent, such as BOP reagent. Protect the 2-amino
substituent of
compounds (v) with a tert-butoxycarbonyl group. Hydrolyze the resulting
compounds (vi) with
reagents such as LiOH in a mixture of THF and methanol to afford compounds
(vii). Convert the
C-8 carboxylic acid of (vii) to the amide group using known reagents such as
HBTU and a
tertiary amine base. Acid-mediated deprotection of compounds (viii) using a
reagent such as
TFA in dichloromethane provides the target compounds (ix).
Scheme C2: Alternative Synthesis of C-8 Carboxamides
NHBoc 0 NHBoc
Br N._ CO /XantPhos / Pd(OAc)2 N......
__________________________________________ . HO
--- K3PO4 ----
NRR THF / H20 NRR'
o o
[0543] React (i) under standard conditions used for the carbonylation of aryl
halides such as
carbon monoxide, a palladium catalyst such as Pd(OAc)2 and a ligand such as
4,5-
241
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
bis(diphenylphosphino)-9,9-dimethylxanthene (XantPhos) and a base such as
potassium
phosphate in a mixture of THF and water to provide carboxylic acids (ii).
Conversion to final
products can then be carried out in a manner similar to that described in
Scheme 1 (vii ¨> ix).
Scheme C3: Synthesis of C-8 Amine Analogs
NH2
Br NO2 Br NO2
condensation
CN reductive cyclization
OEt OEt
0 0
NHBoc NHBoc
N__
1. protect Br amide coupling Br N__
halogen-amine
2. saponify
OH NRR exchange
0 0
iv
NHBoc NHBoc NH2
R"X-N
acylate
deptotect
0 0
or
NRR' sulfonylate NRR' NRR'
0
0 0
viia; X=0 viii
vi viib; X=S0
[0544] React an aldehyde (i) with an appropriately Wittig reagent, such as
ethyl 3-cyano-2-
(triphenylphosphorylidene)propanoate, at ambient temperature to afford an
olefin (ii), which
undergoes reductive cyclization by treating the olefin (ii) with a reducing
agent, such as iron
powder in hot acetic acid, to afford azepines (iii). Protect the C-2 amine
group by using Boc
anhydride to give compounds (iii), which is in turn saponified with an
alkaline metal hydroxide
such as LiOH to afford the carboxylic acid which is coupled with a substituted
amine using a
coupling agent, such as BOP reagent to provide compounds (iv). Convert the C-8
carboxylic
acid of (v) to the amide group using known reagents such as EDCI / HOBT and a
tertiary amine
base. Halogen-amine exchange can be effected using standard methodology such
as copper-
mediated or palladium-catalyzed couplings (benzophenone imine / Pd(II)) to
provide C-8 anilines
(vi). Functionalization of amines (vi) by acylation or sulfonylation provides
anilides (X,C) or
sulfonamides (X=S0) compounds (vii). Alternatively, compounds (vii) can be
prepared directly
through a palladium-mediated coupling of bromide (v) and an appropriately
substituted amide or
sulfonamide. Acid-mediated deprotection of compounds (vii) using a reagent
such as TFA in
dichloromethane provides the target compounds (viii).
242
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Scheme C4: Synthesis of C-8 Sulfur Analogs
NHBoc NHBoc
PHCH2SH
Br S 1. DCDMH
Pd2(dba) 3 / XantPhos 2. R"'NH2
NRR'
dioxane / heat NRR'
0 0
NHBoc 02 NHBoc
02
,S
TFA
NRR'
NRR'
0
0
iv
[0545] React (i) with benzyl thiol in the presence of a palladium catalyst
such as Pd2(dba)3 and a
ligand such as XantPhos at elevated temperatures to provide C-8 sulfides (ii).
Oxidative
chlorination of sulfides (ii) with a reagent such as 1,3-dichloro-5,5-
dimethylhydantoin (DCDMH)
affords intermediate sulfonyl chlorides which can be reacted with an
appropriately substituted
amine of structure R"NH2 to provides sulfonamides (iii). Acid-mediated
deprotection of
compounds (iii) using a reagent such as TFA in dichloromethane provides the
target compounds
(iv).
[0546] These conjugates can be made by various methods. It is understood that
one skilled in the
art may be able to make these compounds by similar methods or by combining
other methods
known to one skilled in the art. It is also understood that one skilled in the
art would be able to
make, in a similar manner as described herein by using the appropriate
starting materials and
modifying the synthetic route as needed. Starting materials and reagents can
be obtained from
commercial vendors or synthesized according to sources known to those skilled
in the art or
prepared as described herein.
[0547] In some aspects, a conjugate comprises: a) an antibody construct
comprising: i) first
binding domain, wherein the first binding domain specifically binds to a tumor
antigen; ii) a
second binding domain, wherein the second binding domain specifically binds to
an antigen on
an antigen presenting cell, wherein the antigen is a molecule on the antigen
presenting cell; and
iii) an Fc domain; b) an immune-stimulatory compound; and c) a linker
attaching the antibody
construct to the immune-stimulatory compound, wherein the linker is covalently
bound to the
antibody construct and the linker is covalently bound to the immune-
stimulatory compound, and
wherein a molar ratio of immune-stimulatory compound to antibody construct is
less than 8;
wherein the first binding domain is attached to the Fc domain and the second
binding domain is
243
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
attached to the Fc domain or to a C-terminal end of a light chain of the first
binding domain;
wherein a Kd for binding of the Fc domain to an Fc receptor in a presence of
the first binding
domain and the second binding domain is no greater than about 100 times a Kd
for binding of the
Fc domain to the Fc receptor in an absence of the second binding domain; and
wherein immune
cell activation caused by the conjugate when bound to the tumor antigen as
measured by a
cytokine release assay is greater than immune cell activation in the absence
of binding to the
tumor antigen.
[0548] In some aspects, a conjugate comprises: a) an antibody construct
comprising: i) first
binding domain, wherein the first binding domain specifically binds to a tumor
antigen; ii) a
second binding domain, wherein the second binding domain specifically binds to
an antigen on
an antigen presenting cell, wherein the antigen is a molecule on the antigen
presenting cell; and
iii) an Fc domain; b) an immune-stimulatory compound; and c) a linker
attaching the antibody
construct to the immune-stimulatory compound, wherein the linker is covalently
bound to the
antibody construct and the linker is covalently bound to the immune-
stimulatory compound, and
wherein a molar ratio of immune-stimulatory compound to antibody construct is
less than 8;
wherein the first binding domain is attached to the Fc domain and the second
binding domain is
attached to the Fc domain or to a C-terminal end of a light chain of the first
binding domain;
wherein a Kd for binding of the Fc domain to an Fc receptor in a presence of
the first binding
domain and the second binding domain is no greater than about 100 times a Kd
for binding of the
Fc domain to the Fc receptor in an absence of the second binding domain;
andwherein antigen
presenting cells are conditionally activated when the conjugate is bound to
the tumor antigen as
measured by a cytokine release assay.
In some aspects, a conjugate for use in inducing immune cell activation
comprising: a) an
antibody construct comprising: i) first binding domain, wherein the first
binding domain
specifically binds to a tumor antigen; ii) a second binding domain, wherein
the second binding
domain specifically binds to an antigen on an antigen presenting cell, wherein
the antigen is a
molecule on the antigen presenting cell; and iii) an Fc domain; b) an immune-
stimulatory
compound; and c) a linker attaching the antibody construct to the immune-
stimulatory compound,
wherein the linker is covalently bound to the antibody construct and the
linker is covalently
bound to the immune-stimulatory compound, and wherein a molar ratio of immune-
stimulatory
compound to antibody construct is less than 8; wherein the first binding
domain is attached to the
Fc domain and the second binding domain is attached to the Fc domain or to a C-
terminal end of
a light chain of the first binding domain; wherein a Kd for binding of the Fc
domain to an Fc
receptor in a presence of the first binding domain and the second binding
domain is no greater
244
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
than about 100 times a Kd for binding of the Fc domain to the Fc receptor in
an absence of the
second binding domain; and wherein immune cell activation caused by the
conjugate when
bound to the tumor antigen as measured by a cytokine release assay is greater
than immune cell
activation in the absence of binding to the tumor antigen.
[0549] In some aspects, a conjugate for use in conditionally activating an
antigen presenting cell
comprising: a) an antibody construct comprising: i) first binding domain,
wherein the first
binding domain specifically binds to a tumor antigen; ii) a second binding
domain, wherein the
second binding domain specifically binds to an antigen on the antigen
presenting cell, and iii) an
Fc domain; b) an immune-stimulatory compound; and c) a linker attaching the
antibody construct
to the immune-stimulatory compound, wherein the linker is covalently bound to
the antibody
construct and the linker is covalently bound to the immune-stimulatory
compound, and wherein a
molar ratio of immune-stimulatory compound to antibody construct is less than
8; wherein the
first binding domain is attached to the Fc domain and the second binding
domain is attached to
the Fc domain or to a C-terminal end of a light chain of the first binding
domain; wherein a Kd
for binding of the Fc domain to an Fc receptor in a presence of the first
binding domain and the
second binding domain is no greater than about 100 times a Kd for binding of
the Fc domain to
the Fc receptor in an absence of the second binding domain; and wherein
antigen presenting cells
are conditionally activated when the conjugate is bound to the tumor antigen
as measured
cytokine release assay.
[0550] In some aspects, a conjugate comprises: a) an immune-stimulatory
compound; b) an
antibody construct comprising a first binding domain and an Fc domain, wherein
the first binding
domain specifically binds to an antigen expressed on a cell, wherein the amino
acid sequence of
the antigen has at least 80% homology to the amino acid sequence of an antigen
selected from a
group consisting of Endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6,
CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, LRRC15, DEC-205,
CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, 41BBL,
MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A, CD16A, HVEM, and CD32B, and a
fragment thereof; and c) a linker attaching the antibody construct to the
immune-stimulatory
compound, wherein the linker is covalently bound to the antibody construct and
the linker is
covalently bound to the immune-stimulatory compound, and wherein a molar ratio
of immune-
stimulatory compound to antibody construct is less than 8.
[0551] In some aspects, a conjugate comprises: a) an immune-stimulatory
compound; b) an
antibody construct comprising a first binding domain and an Fc domain,
wherein: i) the first
binding domain specifically binds to an antigen, wherein the amino acid
sequence of the antigen
245
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
has at least 80% homology to the amino acid sequence of an antigen selected
from a group
consisting of endoglin, PD-L1, CD204, CD206, CD301, VTCN1, VISTA, GLP-3,
CLDN6,
CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorf186, LRRC15, DEC-205,
CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN, CLEC12A, BDCA-2, OX4OL,
41BBL, MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A, CD16A, HVEM, CD32B, and
CD47, and a fragment thereof, ii) a Kd for binding of the first binding domain
to the antigen in a
presence of the immune-stimulatory compound is less than about 100 nM and no
greater than
about 100 times a Kd for binding of the first binding domain to the antigen in
the absence of the
immune-stimulatory compound, and iii) a Kd for binding of the Fc domain to an
Fc receptor in
the presence of the immune-stimulatory compound is no greater than about 100
times a Kd for
binding of the Fc domain to the Fc receptor in the absence of the immune-
stimulatory compound;
and c) a linker attaching the antibody construct to the immune-stimulatory
compound, wherein
the linker is covalently bound to the antibody construct and the linker is
covalently bound to the
immune-stimulatory compound, and wherein a molar ratio of immune-stimulatory
compound to
antibody construct is less than 8.
[0552] In some aspects, a conjugate comprises: a) an immune-stimulatory
compound; b) an
antibody construct comprising a first binding domain and an Fc domain,
wherein: i) the first
binding domain comprises a variable region comprising a set of CDR sequences
that comprises at
least 80% sequence identity to a set of variable region CDR sequences set
forth in TABLE 3 or
TABLE 11; ii) a Kd for binding of the first binding domain to the antigen in a
presence of the
immune-stimulatory compound is less than about 100 nM and no greater than
about 100 times a
Kd for binding of the first binding domain to the antigen in the absence of
the immune-
stimulatory compound, and iii) a Kd for binding of the Fc domain to an Fc
receptor in the
presence of the immune-stimulatory compound is no greater than about 100 times
a Kd for
binding of the Fc domain to the Fc receptor in the absence of the immune
stimulatory compound;
and c) a linker attaching the antibody construct to the immune-stimulatory
compound, wherein
the linker is covalently bound to the antibody construct and the linker is
covalently bound to the
immune-stimulatory compound, and wherein a molar ratio of immune-stimulatory
compound to
antibody construct is less than 8.
[0553] In some aspects, a conjugate for use in activating an immune cell
comprises: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein the first binding domain specifically binds to an
antigen expressed on a
cell, wherein the amino acid sequence of the antigen has at least 80% homology
to the amino
acid sequence of an antigen selected from a group consisting of Endoglin,
CD204, CD206,
246
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4,
TMEM238, Clorf186, LRRC15, DEC-205, CD36 mannose scavenger receptor 1, CLEC9A,
DC-
SIGN, CLEC12A, BDCA-2, 41BBL, MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A,
CD16A, HVEM, and CD32B, and a fragment thereof; and c) a linker attaching the
antibody
construct to the immune-stimulatory compound, wherein the linker is covalently
bound to the
antibody construct and the linker is covalently bound to the immune-
stimulatory compound, and
wherein a molar ratio of immune-stimulatory compound to antibody construct is
less than 8; and
wherein immune cell activation caused by the conjugate when bound to the tumor
antigen as
measured by a cytokine release assay is greater than immune cell activation is
greater than
immune cell activation in the absence of binding to the tumor antigen.
[0554] In some aspects, a conjugate for use in activating an immune cell
comprises: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein: i) the first binding domain specifically binds to an
antigen, wherein the
amino acid sequence of the antigen has at least 80% homology to the amino acid
sequence of an
antigen selected from a group consisting of endoglin, PD-L1, CD204, CD206,
CD301, VTCN1,
VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238,
Clorf186, LRRC15, DEC-205, CD36 mannose scavenger receptor 1, CLEC9A, DC-SIGN,
CLEC12A, BDCA-2, OX4OL, 41BBL, MARCO, CLEC5A, Dectin 1, Dectin 2, CD64, CD32A,
CD16A, HVEM, CD32B, and CD47, and a fragment thereof, ii) a Kd for binding of
the first
binding domain to the antigen in a presence of the immune-stimulatory compound
is less than
about 100 nM and no greater than about 100 times a Kd for binding of the first
binding domain to
the antigen in the absence of the immune-stimulatory compound, and iii) a Kd
for binding of the
Fc domain to an Fc receptor in the presence of the immune-stimulatory compound
is no greater
than about 100 times a Kd for binding of the Fc domain to the Fc receptor in
the absence of the
immune-stimulatory compound; and c) a linker attaching the antibody construct
to the immune-
stimulatory compound, wherein the linker is covalently bound to the antibody
construct and the
linker is covalently bound to the immune-stimulatory compound, and wherein a
molar ratio of
immune-stimulatory compound to antibody construct is less than 8; and wherein
immune cell
activation caused by the conjugate when bound to the tumor antigen as measured
by a cytokine
release assay is greater than immune cell activation is greater than immune
cell activation in the
absence of binding to the tumor antigen.
[0555] In some aspects, a conjugate for use in activating an immune cell
comprises: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein the first binding domain comprises a variable region
comprising a set of
247
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
CDR sequences that comprises at least 80% sequence identity to a set of
variable region CDR
sequences set forth in TABLE 3 or TABLE 11; c) a linker attaching the antibody
construct to
the immune-stimulatory compound, wherein the linker is covalently bound to the
antibody
construct and the linker is covalently bound to the immune-stimulatory
compound, and wherein a
molar ratio of immune-stimulatory compound to antibody construct is less than
8; and wherein
immune cell activation caused by the conjugate when bound to the tumor antigen
as measured by
a cytokine release assay is greater than immune cell activation is greater
than immune cell
activation in the absence of binding to the tumor antigen.
[0556] In some aspects, a conjugate for use in activating an immune cell
comprising: a) an
immune-stimulatory compound; b) an antibody construct comprising a first
binding domain and
an Fc domain, wherein: i) the first binding domain comprises a variable region
comprising a set
of CDR sequences that comprises at least 80% sequence identity to a set of
variable region CDR
sequences set forth in TABLE 3 or TABLE 11; ii) a Kd for binding of the first
binding domain
to the antigen in a presence of the immune-stimulatory compound is less than
about 100 nM and
no greater than about 100 times a Kd for binding of the first binding domain
to the antigen in the
absence of the immune-stimulatory compound, and iii) a Kd for binding of the
Fc domain to an
Fc receptor in the presence of the immune-stimulatory compound is no greater
than about 100
times a Kd for binding of the Fc domain to the Fc receptor in the absence of
the immune
stimulatory compound; and c) a linker attaching the antibody construct to the
immune-
stimulatory compound, wherein the linker is covalently bound to the antibody
construct and the
linker is covalently bound to the immune-stimulatory compound, and wherein a
molar ratio of
immune-stimulatory compound to antibody construct is less than 8; and wherein
immune cell
activation caused by the conjugate when bound to the tumor antigen as measured
by a cytokine
release assay is greater than immune cell activation is greater than immune
cell activation in the
absence of binding to the tumor antigen. In some embodiments, the first
binding domain
comprises a variable region comprising VH and VL sequences at least 80%
sequence identity to a
pair of VH and VL sequences set forth in TABLE 5 or TABLE 13.
Pharmaceutical Formulations
[0557] The conjugates, antibody constructs, recombinant bispecific antibodies
and methods
described herein can be considered useful as pharmaceutical compositions for
administration to a
subject in need thereof. As used herein the terms "individual," "subject," and
"patient" are used
interchangeably and include humans diagnosed with or suspected of being
afflicted with a tumor,
a cancer or other neoplasm. As will be appreciated by the skilled artisan, the
following
248
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
description of pharmaceutical formulations of conjugates and antibody
constructs is applicable to
recombinant bispecific antibodies and conjugates comprising recombinant
bispecific antibodies.
[0558] Pharmaceutical compositions can comprise at least the antibody
constructs and/or
conjugates described herein and one or more pharmaceutically acceptable
carriers, diluents,
excipients, stabilizers, dispersing agents, suspending agents, and/or
thickening agents. The
pharmaceutical composition can comprise the antibody construct. The
pharmaceutical
composition can comprise the conjugate comprising an antibody construct and an
immune-
stimulatory compound, such as an agonist. The pharmaceutical composition can
comprise the
conjugate comprising an Fc domain, a binding domain, and an immune-stimulatory
compound,
such as an agonist. The pharmaceutical composition can comprise any conjugate
described herein.
The pharmaceutical composition can contain a anti-CD40 antibody. A
pharmaceutical
composition can comprise an anti-CD40 antibody conjugated to PAMP molecule. A
pharmaceutical composition can comprise an anti-CD40 antibody conjugated to a
DAMP
molecule. A pharmaceutical composition can further comprise buffers,
antibiotics, steroids,
carbohydrates, drugs (e.g., chemotherapy drugs), radiation, polypeptides,
chelators, adjuvants
and/or preservatives.
[0559] In a pharmaceutical composition, the conjugates can have an average
drug loading. The
drug loading, p, is the average number of immune-stimulatory compound-linker
molecules per
antibody construct, or the number of immune-stimulatory compounds per antibody
construct. P
can range ranges from 1 to 20, or 1-100. In some conjugates, p is preferably
from 1 to 8. The
average number of immune-stimulatory compounds per antibody construct in a
preparation may
be characterized by conventional means such as mass spectroscopy, HIC, ELISA
assay, and
HPLC.
[0560] Pharmaceutical compositions can be formulated using one or more
physiologically-
acceptable carriers comprising excipients and auxiliaries. Formulation can be
modified
depending upon the route of administration chosen. Pharmaceutical compositions
comprising a
conjugate as described herein can be manufactured, for example, by
lyophilizing the conjugate,
mixing, dissolving, emulsifying, encapsulating or entrapping the conjugate.
Pharmaceutical
compositions comprising an antibody construct as described herein can be
manufactured, for
example, by lyophilizing the antibody construct, mixing, dissolving,
emulsifying, encapsulating
or entrapping the antibody construct. The pharmaceutical compositions can also
include the
conjugates or antibody constructs described herein in a free-base form or
pharmaceutically-
acceptable salt form.
249
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0561] Methods for formulation of the pharmaceutical compositions can include
formulating any
of the conjugates or antibody constructs described herein with one or more
inert,
pharmaceutically-acceptable excipients or carriers to form a solid, semi-
solid, or liquid
composition. Solid compositions can include, for example, powders, tablets,
dispersible granules
and capsules, and in some aspects, the solid compositions further contain
nontoxic, auxiliary
substances, for example wetting or emulsifying agents, pH buffering agents,
and other
pharmaceutically-acceptable additives. Alternatively, the compositions
described herein can be
lyophilized or in powder form for re-constitution with a suitable vehicle,
e.g., sterile pyrogen-free
water, before use
[0562] Pharmaceutical compositions of the conjugates or antibody constructs
described herein
can comprise at least a conjugate or antibody construct as an active
ingredient, respectively. The
active ingredients can be entrapped in microcapsules prepared, for example, by
coacervation
techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or
gelatin
microcapsules and poly-(methylmethacylate) microcapsules, respectively), in
colloidal drug-
delivery systems (e.g., liposomes, albumin micro spheres, microemulsions, nano-
particles and
nanocapsules) or in macroemulsions.
[0563] Pharmaceutical compositions as described herein often further can
comprise more than
one active compound as necessary for the particular indication being treated.
The active
compounds can have complementary activities that do not adversely affect each
other. For
example, the pharmaceutical composition can also comprise a chemotherapeutic
agent, cytotoxic
agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic
agent, and/or
cardioprotectant. Such molecules can be present in combination in amounts that
are effective for
the purpose intended.
[0564] The pharmaceutical compositions and formulations can be sterilized.
Sterilization can be
accomplished by filtration through sterile filtration.
[0565] The pharmaceutical compositions described herein can be formulated for
administration
as an injection. Non-limiting examples of formulations for injection can
include a sterile
suspension, solution or emulsion in oily or aqueous vehicles. Suitable oily
vehicles can include,
but are not limited to, lipophilic solvents or vehicles such as fatty oils or
synthetic fatty acid
esters, or liposomes. Aqueous injection suspensions can contain substances
which increase the
viscosity of the suspension. The suspension can also contain suitable
stabilizers. Injections can be
formulated for bolus injection or continuous infusion. Alternatively, the
pharmaceutical
compositions described herein can be lyophilized or in powder form for
reconstitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
250
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0566] For parenteral administration, the recombinant antibodies and
conjugates can be
formulated in a unit dosage injectable form (e.g., use letter solution,
suspension, emulsion) in
association with a pharmaceutically acceptable parenteral vehicle. Such
vehicles can be
inherently nontoxic, and non-therapeutic. A vehicle can be water, saline,
Ringer's solution,
dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as
fixed oils and
ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle
can contain minor
amounts of additives such as substances that enhance isotonicity and chemical
stability (e.g.,
buffers and preservatives).
[0567] Sustained-release preparations can also be prepared. Examples of
sustained-release
preparations can include semipermeable matrices of solid hydrophobic polymers
that can contain
the antibody, and these matrices can be in the form of shaped articles (e.g.,
films or
microcapsules). Examples of sustained-release matrices can include polyesters,
hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,
copolymers of L-
glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic
acid-glycolic acid copolymers such as the LUPRON DEPOTM (i.e., injectable
microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-( ¨ )-3-
hydroxybutyric acid.
[0568] Pharmaceutical formulations of the compositions described herein can be
prepared for
storage by mixing a conjugate or antibody construct with a pharmaceutically
acceptable carrier,
excipient, and/or a stabilizer. This formulation can be a lyophilized
formulation or an aqueous
solution. Acceptable carriers, excipients, and/or stabilizers can be nontoxic
to recipients at the
dosages and concentrations used. Acceptable carriers, excipients, and/or
stabilizers can include
buffers such as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid
and methionine; preservatives, polypeptides; proteins, such as serum albumin
or gelatin;
hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other
carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium;
metal complexes;
and/or non-ionic surfactants or polyethylene glycol.
[0569] In certain embodiments, the recombinant antibodies and conjugates of
the current
disclosure are administered in or packaged with a pharmaceutically acceptable
carrier. The
antibodies can be administered suspended in a sterile solution. In certain
embodiments, the
solution comprises 0.9% NaCl. In certain embodiments, the solution further
comprises one or
more of: buffers, for example, acetate, citrate, histidine, succinate,
phosphate, bicarbonate and
hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80
(Tween 80),
251
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
polysorbate 20 (Tween 20), and poloxamer 188;
polyol/disaccharide/polysaccharides, for
example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose,
or dextran 40; amino
acids, for example, glycine or arginine; antioxidants, for example, ascorbic
acid, methionine; and
chelating agents, for example, EGTA or EGTA. In certain embodiments, the
antibodies and
conjugates of the current disclosure are shipped/stored lyophilized and
reconstituted before
administration. In certain embodiments, lyophilized antibody formulations
comprise a bulking
agent such as mannitol, sorbitol, sucrose, trehalose, or dextran 40.
[0570]
Therapeutic Applications
[0571] The conjugates, antibody constructs, recombinant bispecific antibodies
and
pharmaceutical compositions thereof, and methods of the present disclosure can
be useful for a
plurality of different subjects including, but are not limited to, a mammal,
human, non-human
mammal, a domesticated animal (e.g., laboratory animals, household pets, or
livestock), non-
domesticated animal (e.g., wildlife), dog, cat, rodent, mouse, hamster, cow,
bird, chicken, fish,
pig, horse, goat, sheep, rabbit, and any combination thereof. As will be
appreciated by the skilled
artisan, the following description of therapeutic applications of antibody
constructs and
conjugates comprising antibody constructs is applicable to recombinant
bispecific antibodies and
conjugates comprising recombinant bispecific antibodies.
[0572] The conjugates, antibody constructs, pharmaceutical compositions, and
methods
described herein can be useful as a therapeutic, for example a treatment that
can be administered
to a subject in need thereof. A therapeutic effect can be obtained in a
subject by reduction,
suppression, remission, or eradication of a disease state, including, but not
limited to, a symptom
thereof. A therapeutic effect in a subject having a disease or condition, or
pre-disposed to have or
is beginning to have the disease or condition, can be obtained by a reduction,
a suppression, a
prevention, a remission, or an eradication of the condition or disease, or pre-
condition or pre-
disease state.
[0573] In practicing the methods described herein, therapeutically-effective
amounts of the
conjugates, antibody constructs, or pharmaceutical compositions described
herein can be
administered to a subject in need thereof, often for treating and/or
preventing a condition or
progression thereof. A pharmaceutical composition can affect the physiology of
the subject, such
as the immune system, inflammatory response, or other physiologic affect. A
therapeutically-
effective amount can vary widely depending on the severity of the disease, the
age and relative
health of the subject, the potency of the compounds used, and other factors.
252
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0574] Treat and/or treating can refer to any indicia of success in the
treatment or amelioration of
the disease or condition. Treating can include, for example, reducing,
delaying or alleviating the
severity of one or more symptoms of the disease or condition, or it can
include reducing the
frequency with which symptoms of a disease, defect, disorder, or adverse
condition, and the like,
are experienced by a patient. Treat can be used herein to refer to a method
that results in some
level of treatment or amelioration of the disease or condition, and can
contemplate a range of
results directed to that end, including but not restricted to prevention of
the condition entirely.
[0575] Prevent, preventing and the like can refer to the prevention of the
disease or condition,
e.g., tumor formation, in the patient. For example, if an individual at risk
of developing a tumor
or other form of cancer is treated with the methods of the present disclosure
and does not later
develop the tumor or other form of cancer, then the disease has been
prevented, at least over a
period of time, in that individual.
[0576] Prevent, preventing and the like also can refer to the prevention of
relapse of the disease
or condition, e.g., tumor formation, in the patient. For example, an
individual at risk of relapse of
a tumor or other form of cancer after treatment and obtaining a state of
remission can be treated
with the methods of the present disclosure to present relapse.
[0577] A therapeutically effective amount can be the amount of conjugates,
antibody constructs,
or pharmaceutical compositions or an active component thereof sufficient to
provide a beneficial
effect or to otherwise reduce a detrimental non-beneficial event to the
individual to whom the
composition is administered. A therapeutically effective dose can be a dose
that produces one or
more desired or desirable (e.g., beneficial) effects for which it is
administered, such
administration occurring one or more times over a given period of time. An
exact dose can
depend on the purpose of the treatment, and can be ascertainable by one
skilled in the art using
known techniques.
[0578] The conjugates, antibody constructs, or pharmaceutical compositions
described herein
that can be used in therapy can be formulated and dosages established in a
fashion consistent
with good medical practice taking into account the disorder to be treated, the
condition of the
individual patient, the site of delivery of the conjugate, antibody construct,
or pharmaceutical
composition, the method of administration and other factors known to
practitioners. The
conjugates, antibody constructs, or pharmaceutical compositions can be
prepared according to
the description of preparation described herein.
[0579] One of ordinary skill in the art would understand that the amount,
duration and frequency
of administration of a pharmaceutical composition, antibody construct, or
conjugate described
herein to a subject in need thereof depends on several factors including, for
example but not
253
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
limited to, the health of the subject, the specific disease or condition of
the patient, the grade or
level of a specific disease or condition of the patient, the additional
therapeutics the subject is
being or has been administered, and the like.
[0580] The methods, conjugates, antibody constructs, and pharmaceutical
compositions
described herein can be for administration to a subject in need thereof.
Often, administration of
the conjugates, recombinant antibodies, antibody constructs, or pharmaceutical
compositions can
include routes of administration, non-limiting examples of administration
routes include
intravenous, intraarterial, subcutaneous, subdural, intramuscular,
intracranial, intrasternal,
intratumoral, or intraperitoneally. Additionally, a pharmaceutical
composition, antibody
construct, or conjugate can be administered to a subject by additional routes
of administration,
for example, by inhalation, oral, dermal, intranasal, or intrathecal
administration.
[0581] Pharmaceutical compositions, antibody constructs, or conjugates of the
present disclosure
can be administered to a subject in need thereof in a first administration,
and in one or more
additional administrations. The one or more additional administrations can be
administered to the
subject in need thereof minutes, hours, days, weeks or months following the
first administration.
Any one of the additional administrations can be administered to the subject
in need thereof less
than 21 days, or less than 14 days, less than 10 days, less than 7 days, less
than 4 days or less
than 1 day after the first administration. The one or more administrations can
occur more than
once per day, more than once per week or more than once per month. The
conjugates, antibody
constructs, or pharmaceutical compositions can be administered to the subject
in need thereof in
cycles of 21 days, 14 days, 10 days, 7 days, 4 days or daily over a period of
one to seven days.
[0582] In some aspects, a pharmaceutical composition comprises any recombinant
bispecific
antibody as described herein and a pharmaceutically acceptable carrier.
[0583] In some aspects, a pharmaceutical composition comprises the conjugate
or antibody
construct of as described herein and a pharmaceutically acceptable carrier.
Increased dosages and reduced side-effects
[0584] In certain embodiments, the conjugates described herein can be
administered in a dosage
that is about 10%, about 25%, about 50%, about 100% or greater than an
antibody from which
one of the tumor antigen binding domains or binding domains that bind to a
molecule on a target
cell, such as a tumor antigen, or on an immune cell, such as an antigen
presenting cell (APC), is
derived. For example, a common regimen for administering pertuzumab comprises
840 mg
intravenous (IV) administered as an initial dose over 60 minutes, followed
every 3 weeks
thereafter by 420 mg IV over 30 to 60 minutes. Using a conjugate of this
disclosure, an initial
dosage can range from 900 mg to 1700 mg or more and a maintenance dose can
range from 450
254
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
mg to 900 mg or more. An increased initial dose and/or maintenance dose can be
used with an
conjugate of this disclosure, such as a bispecific tumor targeting conjugate
comprising a tumor
antigen binding domain that binds to a tumor antigen with at least 80%
homology to the amino
acid sequence of HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6,
NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA,
DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, LRRC15, GLP-3, or a fragment
thereof, as
compared with the respective parental monoclonal antibody. An increased
initial dose and/or
maintenance dose also can be used with any bispecific tumor targeting
conjugate of this
disclosure, such as bispecific tumor targeting conjugate comprising a tumor
antigen binding
domain that binds to a tumor antigen with at least 80% homology to the amino
acid sequence of
LRRC15, GLP-3, CLDN6, CLDN16, UPK1B, VTCN1 and STRA6, or a fragment thereof,
as
compared with the respective parental monoclonal antibody. Antibodies that
bind costimulatory
molecules or other cell surface molecules on immune cells such as APCs can
have small
therapeutic windows and high dose-limiting toxicity. For example, the antibody
CP-870,893 can
be shown to have a maximum tolerated dosage of 0.2 mg/kg to 0.3 mg/kg. Using a
bispecific
tumor targeting conjugate of this disclosure can allow administration of the
conjugate at greater
than 0.2 mg/kg to 0.3 mg/kg that comprises a binding domain derived from CP-
870,893 or any
binding domain that binds to a molecule on an immune cell with at least 80%
homology to the
amino acid sequence of CD40, DEC-205, CD36 mannose scavenger receptor 1, DC-
SIGN,
CLEC9A, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin
2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B, CD47, or a fragment
thereof. Using a bispecific tumor targeting conjugate of this disclosure can
allow administration
of the conjugate at greater than 0.2 mg/kg to 0.3 mg/kg that comprises a
binding domain derived
from CP-870,893 or any binding domain that binds to a molecule on an immune
cell with at least
80% homology to the amino acid sequence of CD40, DEC-205, CD36 mannose
scavenger
receptor 1, DC-SIGN, CLEC9A, CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO,
CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1,
CD32B, CD47, TNFR2, TREM2, or a fragment thereof. Using a bispecific tumor
targeting
conjugate of this disclosure can allow administration of the conjugate at
greater than 0.2 mg/kg to
0.3 mg/kg that comprises a binding domain derived from CP-870,893 or any
binding domain that
binds to a molecule on an APC with at least 80% homology to the amino acid
sequence of CD40,
DEC-205, CD36 mannose scavenger receptor 1, DC-SIGN, CLEC9A, CLEC12A, BDCA-2,
OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2, CLEC10A, CD206, CD64,
CD32A, CD16A, HVEM, PD-L1, CD32B, CD47, or a fragment thereof. Using a
bispecific
255
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
tumor targeting conjugate of this disclosure can allow administration of the
conjugate at greater
than 0.2 mg/kg to 0.3 mg/kg that comprises a binding domain derived from CP-
870,893 or any
binding domain that binds to a molecule on an APC with at least 80% homology
to the amino
acid sequence of CD40, DEC-205, CD36 mannose scavenger receptor 1, DC-SIGN,
CLEC9A,
CLEC12A, BDCA-2, OX4OL, 41BBL, CD204, MARCO, CLEC5A, Dectin 1, Dectin 2,
CLEC10A, CD206, CD64, CD32A, CD16A, HVEM, PD-L1, CD32B, CD47, TNFR2, TREM2,
or a fragment thereof. In certain embodiments, using a bispecific tumor
targeting conjugate of
this disclosure can allow administration of a conjugate that comprises a
binding domain that
binds to a molecule on an immune cell, such as an antigen presenting cell, at
greater levels than
an antibody comprising a binding domain that binds to that molecule on an
antigen presenting
cell. In certain embodiments, a bispecific tumor targeting conjugate that
comprises a binding
domain that binds to a molecule on an immune cell, such as an antigen
presenting cell, can be
administered at levels equivalent to that of an antibody from which the
binding domain is
derived. In certain embodiments, the bispecific tumor targeting conjugate can
be administered at
a level higher than the maximum tolerated dose for bispecific tumor targeting
antibody construct
in the absence of a conjugated immune-stimulatory compound. In certain
embodiments, if the
bispecific tumor targeting antibody construct is conjugated to a
chemotherapeutic agent, then the
conjugate can be administered at a level higher than the maximum tolerated
dose for that
chemotherapeutic agent administered in the absence of the chemotherapeutic
agent conjugated to
a bispecific tumor targeting antibody construct. In certain embodiments,
administration of the
bispecific tumor targeting conjugate can be associated with fewer side effects
than an antibody
from which one of binding domains is derived. In certain embodiments,
administration of the
bispecific tumor targeting conjugate can be associated with fewer side effects
than when the
immune-stimulatory compound is conjugated to an antibody from which one of
binding domains
is derived. In certain embodiments, administration of the bispecific tumor
targeting antibody
construct conjugated to a chemotherapeutic agent can be associated with fewer
side effects than
when the chemotherapeutic agent is conjugated to an antibody from which one of
binding
domains is derived.
[0585] In some embodiments described herein is a method of treating a subject
in need thereof,
comprising administering to the individual a therapeutic dose of a recombinant
bispecific
antibody, or the pharmaceutical composition, to an individual having cancer.
In some
embodiments, the administering comprises administering the recombinant
bispecific antibody, or
the pharmaceutical composition, intravenously, cutaneously, subcutaneously, or
injected at a site
of affliction. In some embodiments, the recombinant bispecific antibody
induces more potent
256
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
immune activation to cancer cells or tissue as compared to non-cancerous
tissue. In some further
embodiments, when the recombinant antibody is administered intraveneously to
the subject at the
minimum anticipated biological effect level of the recombinant antibody, a
biological effect of
the recombinant antibody is greater when the recombinant antibody is bound to
the tumor
associated antigen at the cancer and to the molecule on the antigen presenting
cell as compared to
the biological effect of the recombinant antibody when it is not bound to the
tumor associated
antigen but is bound to the molecule on the antigen presenting cell.
[0586] In additional aspects, the recombinant bispecific antibodies and
conjugates thereof can
have an increased minimum anticipated biological effect level (MABEL), as
compared to a
corresponding mono-specific antibody or conjugate. The MABEL of the
recombinant bispecific
antibody is higher than the MABEL of the control monospecific antibody or
conjugate. When the
recombinant bispecific antibody is administered to a subject at the MABEL, the
biological effect
is greater when the target antigen binding domain is bound to its tumor
associated antigen as
compared to the biological effect of the recombinant antibody when the
recombinant antibody is
not bound to the tumor associated antigen. In some embodiments, the biological
effect is greater
when the target antigen binding domain is bound to its tumor associated
antigen and the effector
antigen binding domain is bound to the molecule on the antigen presenting cell
as compared to
the biological effect of the recombinant antibody when the recombinant
antibody is not bound to
the tumor associated antigen but the effector antigen binding domain is bound
to the molecule on
the antigen presenting cell. The biological effect can be immune activation.
[0587] MABEL is the anticipated dose needed that results in a biological
effect in a human
subject, in which a biological effect is measured by an in vitro, ex vivo,
and/or in vivo assay that
measures a selected biological, biochemical, pharmacological, or
pharmacodynamic effect. A
selected biological, biochemical, pharmacological, or pharmacodynamic effect
can be secretion
of one or more cytokines, secretion of one or more chemokines, expression
level of one or more
cell surface proteins associated with immune stimulation, or activity of one
or more immune cell
functions. For example, the secretion of one or more cytokines can be the
secretion of one or
more of IL-6, TNFa, IL-12p40, IL-12p'70, and/or IL-10. Secretion of one or
more chemokines
can be IL-8, IP-10, MIP-la, and/or MIP-10. Expression level of one or more
cell surface proteins
associated with immune stimulation can be CD54, CD86, CD80, MHC class II,
and/or CD83.
Activity of one or more immune cell functions can be antibody-dependent cell-
mediated
cytotoxicity, antibody dependent cellular phagocytosis, and/or antigen cross-
presentation.
[0588] In some aspects, when dosing intraveneously at the MABEL of the
recombinant antibody,
when the recombinant antibody is bound to the tumor associated antigen the
biological effect can
257
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
be at least two times, five times, or ten times greater than the biological
effect of the recombinant
antibody when the recombinant antibody is not bound to the tumor associated
antigen. The
biological effect can be immune activation.
[0589] In some aspects, method of treating a subject in need thereof,
comprising administering to
the subject a therapeutic dose of any recombinant bispecific antibody as
described herein or the
pharmaceutical composition of any recombinant bispecific antibody as described
herein. In some
embodiments, the subject has cancer.
[0590] In some embodiments, the recombinant bispecific antibody or the
pharmaceutical
composition is administered intravenously, cutaneously, subcutaneously, or
injected at a site of
affliction. In some embodiments, the recombinant bispecific antibody induces
greater immune
activation against a cancer as measured by a decrease in cancer cell number or
volume as
compared to non-cancerous tissue.
[0591] In some embodiments, the recombinant bispecific antibody is
administered intravenously
to the subject at a minimum anticipated biological effect level of the
recombinant bispecific
antibody, a biological effect of the recombinant bispecific antibody is
greater when the
recombinant bispecific antibody is bound to the tumor associated antigen and
to the antigen on
the antigen presenting cell as compared to the biological effect of the
recombinant bispecific
antibody when it is not bound to the tumor associated antigen but is bound to
the antigen on the
antigen presenting cell; and wherein the biological effect is immune
activation as measured by
one or more of the group selected from secretion of one or more cytokines,
secretion of one or
more chemokines, expression level of one or more cell surface proteins
associated with immune
stimulation, antibody-dependent cell-mediated cytotoxicity, antibody dependent
cellular
phagocytosis, and antigen cross-presentation.
[0592] In some embodiments, the recombinant bispecific antibody is
administered intravenously
to the subject at the minimum anticipated biological effect level of the
recombinant bispecific
antibody, it induces a greater biological effect at the site of the cancer
than at a non-cancerous
site and wherein the biological effect is immune activation as measured by one
or more of the
group selected from secretion of one or more cytokines, secretion of one or
more chemokines,
expression level of one or more cell surface proteins associated with immune
stimulation,
antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular
phagocytosis, and
antigen cross-presentation.
Additional therapeutic agents
[0593] In certain embodiments, conjugates, antibody constructs, recombinant
bispecific
antibodies, pharmaceutical compositions thereof, and methods provided herein
can be
258
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
administered with or during treatment with an additional therapeutic agent. As
will be
appreciated by the skilled artisan, the following description as applied to
antibody constructs and
conjugates comprising antibody constructs is applicable to recombinant
bispecific antibodies and
conjugates comprising recombinant bispecific antibodies
[0594] In certain embodiments, the therapeutic agent comprises a recombinant
protein or
monoclonal antibody. In certain embodiments, the recombinant protein or
monoclonal antibody
comprises Etaracizumab (Abegrin), Tacatuzumab tetraxetan, Bevacizumab
(Avastin),
Labetuzumab, Cetuximab (Erbitux), Obinutuzumab (Gazyva), Trastuzumab
(Herceptin),
Clivatuzumab, Trastuzumab emtansine (Kadcyla), Rituximab (MabThera, Rituxan),
Gemtuzumab ozogamicin (Mylotarg), Girentuximab (Rencarex), or Nimotuzumab
(Theracim,
Theraloc). In certain embodiments, the additional therapeutic agent is a
chemotherapeutic agent.
In certain embodiments, the chemotherapeutic agent is an alkylating agent
(e.g.,
cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,
mechlorethamine,
uramustine, thiotepa, nitrosoureas, or temozolomide), an anthracycline (e.g.,
doxorubicin,
adriamycin, daunorubicin, epirubicin, or mitoxantrone), a cytoskeletal
disruptor (e.g., paclitaxel
or docetaxel), a histone deacetylase inhibitor (e.g., vorinostat or
romidepsin), an inhibitor of
topoisomerase (e.g., irinotecan, topotecan, amsacrine, etoposide, or
teniposide), a kinase inhibitor
(e.g., bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, or
vismodegib), a nucleoside analog
or precursor analog (e.g., azacitidine, azathioprine, capecitabine,
cytarabine, fluorouracil,
gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine), a
peptide antibiotic
(e.g., actinomycin or bleomycin), a platinum-based agent (e.g., cisplatin,
oxaloplatin, or
carboplatin), or a plant alkaloid (e.g., vincristine, vinblastine,
vinorelbine, vindesine,
podophyllotoxin, paclitaxel, or docetaxel). In some embodiments, the
chemotherapeutic agent is
a nucleoside analog. In some embodiments, the chemotherapeutic agent is
gemcitabine. In certain
embodiments, the additional therapeutic agent is radiation therapy.
[0595] In some embodiments, the recombinant bispecific antibody further
comprises a
chemotherapeutic compound and a linker, wherein the linker links the
chemotherapeutic
compound to the Fc comprising domain.
[0596] In some embodiments, the chemotherapeutic compound comprises an
alkylating agent, an
anthracycline, a cyto skeletal disruptor, a histone deacetylase inhibitor, an
inhibitor of, a kinase
inhibitor, a nucleoside analog or precursor analog, a peptide antibiotic, a
platinum-based
compound, or a plant alkaloid.
259
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0597] In some aspects, a method of treatment for a subject in need thereof
comprises
administering a therapeutic dose of the antibody construct or conjugate as
described herein or the
pharmaceutical composition as described herein. In some embodiments, the
subject has cancer.
[0598] In some embodiments, the antibody construct or conjugate is
administered intravenously,
cutaneously, subcutaneously, or injected at a site of affliction. In some
embodiments, after
administration of antibody construct or conjugate to the subject, immune cell
activation is
increased in the subject as measured by a secretion of one or more cytokines
as measured by a
cytokine release assay, a secretion of one or more chemokines as measured by
an ELISA
immunoassay, an expression level of one or more cell surface proteins
associated with immune
stimulation as measured by an ELISA immunoassay, an activity of one or more
immune cell
functions, or combination thereof, as compared to before administration of the
antibody construct
or conjugate to the subject.
[0599] In some embodiments, the activity of one or more immune cell functions
comprises
antibody-dependent cell-mediated cytotoxicity as measured by an ADCC assay,
antibody
dependent cellular phagocytosis as measured by an ADCP assay, or antigen cross-
presentation as
measured by a cross-presentation assay.
[0600] In some embodiments, after administration of the antibody construct or
conjugate to the
subject, tumor cell intracellular signaling is altered in the subject as
compared to tumor cell
intracellular signaling before administration of the antibody construct or
conjugate as measured
by an intracellular signaling assay.
[0601] In some embodiments, the altered tumor cell intracellular signaling
increases tumor
immunogenicity as measured by an immunogencity assay.
[0602] In some aspects, a kit comprising a pharmaceutically acceptable dosage
unit of a
pharmaceutically effective amount of the conjugate or antibody construct as
described herein or
the pharmaceutical composition as described herein.
Diseases, Conditions and the Like
[0603] The conjugates, antibody constructs, recombinant bispecific antibodies,
pharmaceutical
compositions thereof, and methods provided herein can be useful for the
treatment of a plurality
of diseases, conditions, preventing a disease or a condition in a subject or
other therapeutic
applications for subjects in need thereof. As will be appreciated by the
skilled artisan, the
following description as applied to antibody constructs and conjugates
comprising antibody
constructs is applicable to recombinant bispecific antibodies and conjugates
comprising
recombinant bispecific antibodies.
260
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0604] Often the conjugates, antibody constructs, pharmaceutical compositions,
and methods
provided herein can be useful for treatment of hyperplastic conditions,
including but not limited
to, neoplasms, cancers, tumors and the like. A condition, such as a cancer,
can be associated with
expression of a molecule on the cancer cells. Often, the molecule expressed by
the cancer cells
can comprise an extracellular portion capable of recognition by the antibody
portion of the
antibody construct. A molecule expressed by the cancer cells can be a tumor
antigen. An
antibody portion of the conjugate or pharmaceutical composition can recognize
a tumor antigen.
A tumor antigen can include CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45,
CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLA-DR, carcinoembryonic antigen
(CEA), TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-
specific
membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9,
epidermal growth
factor, p185HER2, IL-2 receptor, EGFRVIII (de2-7 EGFR), fibroblast activation
protein,
tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor,
avf33, WT1, LMP2,
HPV E6 E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ES0-1, MelanA/MART1, Ras
mutant,
gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a Sarcoma
translocation
breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK,
androgen
receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1,
mesothelin (MSLN),
PSCA, MAGE Al, MAGE A3, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH,
ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, 0Y-TES1,
Sperm
protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, Page4,
VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4, MAGE
C2, GAGE, EGFR, CMET, HER3, CA6, NAPI2B, TROP2, Claudin-6 (CLDN6), Claudin-16
(CLDN16), CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, Uroplakin-1B (UPK1B), VTCN1
(B7-H4), STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorf186, LIV1, ROR1, Fos-related
antigen 1, VEGFR1, endoglin, VISTA, or a fragment thereof. .
[0605] As described herein, an antigen binding domain portion of the
conjugate, can be
configured to recognize a molecule expressed by a cancer cell, such as for
example, a disease
antigen, tumor antigen or a cancer antigen. Often such antigens are known to
those of ordinary
skill in the art, or newly found to be associated with such a condition, to be
commonly associated
with, and/or, specific to, such conditions. For example, a disease antigen,
tumor antigen or a
cancer antigen is, but is not limited to, CD5, CD19, CD20, CD25, CD37, CD30,
CD33, CD45,
CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLA-DR, carcinoembryonic antigen
(CEA), TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-
specific
membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9,
epidermal growth
261
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
factor, p185HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation
protein,
tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor,
avf33, WT1, LMP2,
HPV E6, HPV E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ES0-1, MelanA/MART1, Ras
mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, a
Sarcoma
translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17,
PAX3, ALK,
androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1,
mesothelin
(MSLN), PSCA, MAGE Al, MAGE A3, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn,
GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-
TESL Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie
3,
Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4,
MAGE C2, GAGE, EGFR, CMET, HER3, CA6, NAPI2B, TROP2, Claudin-6 (CLDN6),
Claudin-16 (CLDN16), CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, Uroplakin-1B
(UPK1B),
VTCN1 (B7-H4), STRA6, TMPRSS3, TMRRSS4, TMEM238, Clorf186, LIV1, ROR1, Fos-
related antigen 1, VEGFR1, endoglin, VISTA, or a fragment thereof.
Additionally, such tumor
antigens can be derived from the following specific conditions and/or families
of conditions,
including but not limited to, cancers such as brain cancers, skin cancers,
lymphomas, sarcomas,
lung cancer, liver cancer, leukemias, uterine cancer, breast cancer, ovarian
cancer, cervical
cancer, bladder cancer, kidney cancer, hemangio sarcomas, bone cancers, blood
cancers, testicular
cancer, prostate cancer, stomach cancer, intestinal cancers, pancreatic
cancer, and other types of
cancers as well as pre-cancerous conditions such as hyperplasia or the like.
[0606] Non-limiting examples of cancers can include Acute lymphoblastic
leukemia (ALL);
Acute myeloid leukemia; Adrenocortical carcinoma; Astrocytoma, childhood
cerebellar or
cerebral; Basal-cell carcinoma; Bladder cancer; Bone tumor,
osteosarcoma/malignant fibrous
histiocytoma; Brain cancer; Brain tumors, such as, cerebellar astrocytoma,
malignant glioma,
ependymoma, medulloblastoma, visual pathway and hypothalamic glioma; Brainstem
glioma;
Breast cancer; Bronchial adenomas/carcinoids; Burkitt's lymphoma; Cerebellar
astrocytoma;
Cervical cancer; Cholangiocarcinoma; Chondrosarcoma; Chronic lymphocytic
leukemia;
Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon
cancer; Cutaneous
T-cell lymphoma; Endometrial cancer; Ependymoma; Esophageal cancer; Eye
cancers, such as,
intraocular melanoma and retinoblastoma; Gallbladder cancer; Glioma; Hairy
cell leukemia;
Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin
lymphoma;
Hypopharyngeal cancer; Islet cell carcinoma (endocrine pancreas); Kaposi
sarcoma; Kidney
cancer (renal cell cancer); Laryngeal cancer; Leukaemia, such as, acute
lymphoblastic, acute
myeloid, chronic lymphocytic, chronic myelogenous and, hairy cell; Lip and
oral cavity cancer;
262
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Liposarcoma; Lung cancer, such as, non-small cell and small cell; Lymphoma,
such as, AIDS-
related, Burkitt; Lymphoma, cutaneous T-Cell, Hodgkin and Non-Hodgkin,
Macroglobulinemia,
Malignant fibrous histiocytoma of bone/osteosarcoma; Melanoma; Merkel cell
cancer;
Mesothelioma; Multiple myeloma/plasma cell neoplasm; Mycosis fungoides;
Myelodysplastic
syndromes; Myelodysplastic/myeloproliferative diseases; Myeloproliferative
disorders, chronic;
Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma;
Neuroblastoma;
Oligodendroglioma; Oropharyngeal cancer; Osteosarcoma/malignant fibrous
histiocytoma of
bone; Ovarian cancer; Pancreatic cancer; Parathyroid cancer; Pharyngeal
cancer;
Pheochromocytoma; Pituitary adenoma; Plasma cell neoplasia; Pleuropulmonary
blastoma;
Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal
pelvis and ureter,
transitional cell cancer; Rhabdomyosarcoma; Salivary gland cancer; Sarcoma,
Ewing family of
tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary
syndrome; Skin cancer
(non-melanoma); Skin carcinoma; Small intestine cancer; Soft tissue sarcoma;
Squamous cell
carcinoma; Squamous neck cancer with occult primary, metastatic; Stomach
cancer; Testicular
cancer; Throat cancer; Thymoma and thymic carcinoma; Thymoma,; Thyroid cancer;
Thyroid
cancer, childhood; Uterine cancer; Vaginal cancer; Waldenstrom
macroglobulinemia; Wilms
tumor and any combination thereof.
[0607] In certain embodiments, the recombinant bispecific antibodies and
conjugates
comprising recombinant bispecific antibodies are useful for the treatment of a
cancer or tumor. In
certain embodiments, the cancer comprises breast, heart, lung, small
intestine, colon, spleen,
kidney, bladder, head, neck, ovarian, prostate, brain, pancreatic, skin, bone,
bone marrow, blood,
thymus, uterine, testicular and liver tumors. In certain embodiments, tumors
which can be treated
with the recombinant bispecific antibodies (including conjugates thereof)
comprise adenoma,
adenocarcinoma, angio sarcoma, astrocytoma, epithelial carcinoma, germinoma,
glioblastoma,
glioma, hemangioendothelioma, hemangio sarcoma, hematoma, hepatoblastoma,
leukemia,
lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma,
retinoblastoma,
rhabdomyosarcoma, sarcoma and/or teratoma. In certain embodiments, the
tumor/cancer is
selected from the group of acral lentiginous melanoma, actinic keratosis,
adenocarcinoma,
adenoid cystic carcinoma, adenomas, adeno sarcoma, adenosquamous carcinoma,
astrocytic
tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland
carcinoma, capillary
carcinoid, carcinoma, carcino sarcoma, cholangiocarcinoma, chondro sarcoma,
cystadenoma,
endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma,
endometrioid
adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia,
gastronoma,
germ line tumors, glioblastoma, glucagonoma, hemangioblastoma,
hemangioendothelioma,
263
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma,
insulinite,
intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive
squamous cell
carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic
leukemia,
lymphocytic leukemia, leiomyo sarcoma, melanoma, malignant melanoma, malignant
mesothelial
tumor, nerve sheath tumor, medulloblastoma, medulloepithelioma, mesothelioma,
mucoepidermoid carcinoma, myeloid leukemia, multiple myeloma, neuroblastoma,
neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian
carcinoma, papillary
serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, prostate
carcinoma,
pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyo sarcoma,
sarcoma, serous
carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue
carcinoma, somatostatin
secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated
carcinoma,
uveal melanoma, verrucous carcinoma, vagina/vulva carcinoma, vipoma, and
Wilm's tumor. In
certain embodiments, the tumor/cancer to be treated with one or more
recombinant bispecific
antibodies (including conjugates thereof) comprise brain cancer, head and neck
cancer, colorectal
carcinoma, acute myeloid leukemia, pre-B-cell acute lymphoblastic leukemia,
bladder cancer,
astrocytoma, preferably grade II, III or IV astrocytoma, glioblastoma,
glioblastoma multiforme,
small cell cancer, and non-small cell cancer, preferably non-small cell lung
cancer, lung
adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate
cancer,
androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and
breast cancer,
preferably breast ductal cancer, and/or breast carcinoma. In certain
embodiments, the cancer
treated with the recombinant bispecific antibodies (including conjugates
thereof) comprises
glioblastoma. In certain embodiments, the cancer treated with one or more
recombinant
bispecific antibodies (including conjugates thereof) comprises pancreatic
cancer. In certain
embodiments, the cancer treated with one or more recombinant bispecific
antibodies (including
conjugates thereof) comprises ovarian cancer. In certain embodiments, the
cancer treated with
one or more recombinant bispecific antibodies (including conjugates thereof)
comprises or lung
cancer.
[0608] In certain embodiments, the recombinant bispecific antibodies
(including conjugates
thereof) can be administered by any route suitable for the administration of
antibody-containing
pharmaceutical compositions, such as, for example, cutaneous, subcutaneous,
intraperitoneal,
intravenous, intramuscular, intratumoral, intracerebral, or at a tumor
afflicted site, etc. In certain
embodiments, the recombinant bispecific antibodies (including conjugates
thereof) are
administered intravenously. In certain embodiments, the recombinant bispecific
antibodies
(including conjugates thereof) are administered on a suitable dosage schedule,
for example,
264
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
weekly, twice weekly, three times weekly, once every two weeks, once every
three weeks,
monthly, twice monthly, three times monthly, etc. The recombinant bispecific
antibodies
(including conjugates thereof) can be administered in any therapeutically
effective amount. In
certain embodiments, the therapeutically acceptable amount is between about
0.1 mg/kg and
about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount
is between about
1 mg/kg and about 40 mg/kg. In certain embodiments, the therapeutically
acceptable amount is
between about 5 mg/kg and about 30 mg/kg. In certain embodiments, the
therapeutically
acceptable amount is between about 1 mg/kg and about 10 mg/kg.
EXAMPLES
[0609] The following examples are included to further describe some
embodiments of the
present disclosure, and should not be used to limit the scope of the
disclosure
EXAMPLE 1
Synthesis of Linkers with Immune-Stimulatory Compounds
[0610] A linker is linked with an immune-stimulatory compound. A linker linked
to an immune-
stimulatory compound is formed to make a linker-immune stimulatory compound
construct
(ATAC). Subsequently, an ATAC is conjugated to an antibody, in which the ATAC
is any one of
ATAC1 ¨ ATAC34 (each of which is described in the below EXAMPLES).
[0611] A linker is linked with an antibody, in which the linker is a PEGylated
linker, a linker
containing a valine-alanine dipeptide, a linker containing a valine-citrulline
dipeptide, or an N-
Maleimidomethylcyclohexane-l-carboxylate (MCC) linker. Subsequently, an immune-
stimulatory compound is conjugated to the linker linked with the antibody or
antibody construct,
in which the immune-stimulatory compound is a TLR agonist, a Nod-like receptor
ligand, a RIG-
Like receptor agonist, a CLR ligand, a CDS ligand, or an inflammasome inducer.
[0612] A linker is linked with an antibody or antibody construct, in which the
linker is a
PEGylated linker, a linker containing a valine-alanine dipeptide, a linker
containing a valine-
citrulline dipeptide, or an N-Maleimidomethylcyclohexane-l-carboxylate (MCC)
linker.
Subsequently, an immune-stimulatory compound is conjugated to the linker
linked with the
antibody or antibody construct, in which the immune-stimulatory compound is
gardiquimod or
an analog of a cyclic dinucleotide.
EXAMPLE 2
Synthesis of ATAC1 and ATAC2
[0613] This example shows the synthesis of pentafluorophenyl 25-(2-amino-3-
pentylquinolin-5-
y1)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC1) and
Perfluorophenyl 3-((4-
265
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)-4-
oxo-
7,10,13,16,19-pentaoxa-3-azadocosan-22-oate (ATAC2).
T FA
NH2
I 1\1
F
H
F 0 01..0 =e=Oc)0,,..r N
0 0
F F
F
ATAC1
Step A: Preparation of Int ATAC1-1
NH2
1 r\j
0 H2N
0 HOy....,..,õ00...---=,,,O.,.....^....0,---.,..Ø,..õ---....ir .N
_____________________________________________________ ..
0 0
0
HCI
NH2
'N
1
H
HO0e-00.70-).rN
0 0
ATAC1-1
[0614] To a 0 C solution containing 271 mg (0.90 mmol) of 5-(5-aminopenty1)-3-
pentylquinolin-
2-amine in 4 mL of DCM was added 435 mg (1.00 mmol) of the NHS ester in 1 mL
of DCM
dropwise over 15 minutes. The reaction mixture was allowed to warm to ambient
temperature
over 19h before it was concentrated and purified by reverse phase
chromatography. Pure
fractions were lyophilized and dissolved in 3 mL of methanol then treated with
1 mL of 4N HC1
in dioxane. The solution was stirred for lh then concentrated to afford the
desired compound as
an HC1 salt which was used directly in the next step.
266
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Step B. Preparation of ATAC1
H CI F
N H2 F 0 OH
I 1\1 F F
F
H ________________________________________________________ ..-
HO,r.....õ,0..õ..---,0,----,,,O...,õ,,=-...0,-..,-0...,...õThiõN
DIC I DM F
0 0
ATAC 1 -1
TFA
N H2
1 1\1
F
H
F F
0 0
F
ATAC 1
[0615] To a stirred solution of 25-(2-amino-3-pentylquinolin-5-y1)-19-oxo-
4,7,10,13,16-
pentaoxa-20 azapentacosanoic acid hydrochloride (130 mg, 0.198 mmol) and
pentafluorophenol
(146 mg, 0.792 mmol) in DMF (2.5 ml) at room temperature was added N,N'-
Diisopropylcarbodiimide (0.186 ml, 1.189 mmol) dropwise. The reaction was
stirred at room
temperature for 18h and then concentrated. The crude product was added to a
100g C18 gold
reverse phase column and was eluted with water/acetonitrile (0.1% TFA) 10-
100%. The fractions
were combined and concentrated then freeze dried to give perfluorophenyl 25-(2-
amino-3-
pentylquinolin-5-y1)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate-2,2,2-
trifluoroacetate
(110 mg, 61.7 % yield) as a clear gum. 1H NMR (DMSO-d6) 8 13.7 (s, 1H), 8.37-
8.35 (m, 3H),
7.78 (t, J=5.5Hz, 1H), 7.63 (t, J=7.5Hz, 1H), 7.53 (d, J=8.5Hz, 1H), 7.31 (d,
J=7.0 Hz, 1H), 3.58
(t, J=6.0Hz, 2H), 3.63-3.43 (m, 20H), 3.04-2.96 (m, 6H), 2.73 (t, J=7.5Hz,
2H), 2.27 (t, J=7.5Hz,
2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J=7.5Hz, 3H). LCMS [M-FH]
= 786.3.
[0616] The following compound in TABLE 21 can be prepared using a method
similar to that
described above for ATAC1.
TABLE 21
Compound Structure IUPAC
M+1
ATAC2 H2N _IV Perfluorophenyl 3-((4-
amino- 800
F
r NI \ =
F N
1-(2-hydroxy-2-
Ai Onõ0.......,,...,,,O,......,,.,,On.,,),N
F 4" F HO methylpropy1)-1H-
F
imidazoI4,5-c]quinolin-2-
yl)methyl)-4-oxo-
7,10,13,16,19-pentaoxa-3-
267
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
azadocosan-22-oate
EXAMPLE 3
Synthesis of ATAC3 and ATAC4
[0617] This example shows the synthesis of pentafluorophenyl 25-(2-amino-3-
pentylquinolin-5-
y1)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC3) and 2,5-
Dioxopyrrolidin-1-y1
3-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo-[4,5-c]quinolin-2-
y1)methyl)-4-oxo-
7,10,13,16,19-pentaoxa-3-azadocosan-22-oate (ATAC4).
HCI
NH2
I N
0 H
_....1.µ\1,01Ø..,.....õ---....0,....--.......õ..0õ........--..Ø.----...õ.-
0..,.......---yN
0 0
0
ATAC3
Step A. Preparation of ATAC3
[0618] To a stirred solution of Int ATAC1-1 (185 mg, 0.282 mmol) and N-
hydroxysuccinimide
(130 mg, 1.128 mmol) in DMF (3 ml) was added N,N'-diisopropylcarbodiimide
(0.221 ml, 1.411
mmol) dropwise and the reaction mixture was stirred at room temperature for 18
hours. The
reaction mixture was filtered and washed with acetonitrile and the filtrate
was evaporated. The
resulting residue was purified by silica gel Silica gel column chromatography
(DCM / Me0H /
HOAc) to give 65 mg of the desired product as the acetic acid salt which was
subsequently
dissolved in 2 mL of DCM and treated with 2M HC1 in diethyl ether. The
solution was stirred for
lh then concentrated and lyophilized to afford the desired compound as the HC1
salt. 1H NMR
(CDC13) 8 15.2 (s, 1H), 8.15 (d, J=7.8Hz, 1H), 7.68 (d, J=7.9Hz, 1H), 7.55 (t,
J=8.1Hz, 1H), 6.55
(bs, 1H), 3.98 (t, J=6.0Hz, 2H), 3.83-3.55 (m, 18H), 3.33-3.22 (m, 2H), 2.95-
2.56 (m, 11H), 2.27
(t, J=7.5Hz, 2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J=7.5Hz, 3H).
LCMS [M+H] =
717.3.
[0619] The following 2,5-Dioxopyrrolidin-1-y13-((4-amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo-[4,5-c]quinolin-2-y1)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-
22-oate
(ATAC4) compound can be prepared using a method similar to that described
above for ATAC3.
268
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
H2N
_N
0 r N \ I
N 1
I.C ).rC)0C)0C).r -N
0 0
0
1-1-0-
ATAC4
1H NMR (CDC13) 8 14.9 (s, 1H), 8.88 (bs, 1H), 8.15 (d, 1H), 7.85 (d, 1H), 7.61
(t, 1H), 7.45 (t,
1H), 4.72 (s, 2H), 3.83 (m, 4H), 3.65-3.45 (m, 18H), 2.90-2.71 (m, 9H), 1.43
(t, J=7.0Hz, 3H),
1.33 (s, 6H). LCMS [M-Ftl] = 731.
EXAMPLE 4
Synthesis of ATAC5, ATAC6 and ATAC7
[0620] This example shows the synthesis of 2,5-dioxopyrrolidin-1-y16-(((S)-1-
(((S)-1-((4-((((5-
(2-amino-3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-2-
yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC5), 2,5-
dioxopyrrolidin-1-y1
7-(((S)-1-(((S)-1-((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-
c]quinolin-2-
y1)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-
methyl-1-
oxobutan-2-y1)amino)-7-oxoheptanoate (ATAC6), and 2,5-dioxopyrrolidin-1-y17-
(((S)-1-(((S)-
1-((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-c]quinolin-2-
y1)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-1-oxobutan-2-y1)amino)-7-oxoheptanoate (ATAC7).
TFA
NH2
0 0 CH3 N
H ii I
11 )(rNN)H.rEll
0 0 H 0 el OriRli
0 II
0
ATAC5
Step A: Preparation of Int ATAC5-1
269
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
NH2
1\1
NO2 1
H2N
0 CH3
>0 N N )).( N =
DIPEA / DCM
0 0 0 OTO
2 HCI
0
HCI
NH2
0 CH3 1\1
HON,yH
0 0 0 101 0 1\1
0
Int ATAC5-1
[0621] A solution of 5-(5-aminopenty1)-3-pentylquinolin-2-amine (300 mg, 1.00
mmol) in 5 mL
DCM was stirred at room temperature under nitrogen for 10 min before tert-
butyladipate-valine-
alanine-para-aminobenzy1-4-nitrophenylcarbonate (tBuAdip-va-PAB-OPNP, 656 mg,
1.00 mmol)
and DIPEA (0.26 ml, 1.5 mmol) in 3 mL of DCM were added and the mixture was
stirred at
room temperature overnight. The mixture was concentrated and purified by
column
chromatography. Clean fractions were combined and evaporated and the residue
was dissolved
in 2 mL of DCM and treated with 2M HC1 in diethyl ether. The solution was
stirred for lh then
concentrated and lyophilized to afford the desired compound Int ATAC5-1 as the
HC1 salt. MS
m/z 761 (M) .
Step B: Preparation of ATAC5
HCI
NH2
0 CH3
HONH
NHS / DIC
0 0 0
0
Int ATAC5-1
TFA
NH2
0 0 CH3
N,yH
0 0 0 le 0,F
0 Ni
0
ATAC5
[0622] To a stirred solution of 6-(((S)-1-(((S)-1-((4-((((5-(2-amino-3-
pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-
1-oxobutan-
270
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
2-yl)amino)-6-oxohexanoic acid hydrochloride (221 mg, 0.282 mmol) and N-
hydroxysuccinimide (130 mg, 1.12 mmol) in DMF (3 ml) was added N,N'-
diisopropylcarbodiimide (0.221 ml, 1.41 mmol) dropwise and the reaction
mixture was stirred at
room temperature for 5h. HPLC indicated some starting material remained so the
reaction was
stirred at ambient temperature overnight. The reaction mixture was filtered
and washed with
acetonitrile. The filtrate was evaporated and the residue was dissolved in
DMSO and purified by
reverse phase chromatography [water/acetonitrile (0.1% TFA)] from 10% followed
by a gradient
from 20 to 80%. Pure fractions were combined to give 2,5-dioxopyrrolidin-1-y16-
(((S)-1-(((S)-1-
((4-((((5-(2-amino-3-pentylquinolin-5-yl)penty1)-
carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-y1)amino)-6-oxohexanoate 2,2,2-
trifluoroacetate
(109 mg, 40 % yield) as a white solid. 1H NMR (DMSO-d6) 8 13.6 (s, 1H), 9.92
(s, 1H), 8.32
(d, J=7.5Hz, 1H), 7.84 (d, J=8.5Hz,1H), 7.61-7.55 (m, 4H), 7.30-7.17 (m, 4H),
4.92 (s, 2H), 4.37
(t, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 2.96 (m, 4H), 2.81-2.62 (m, 8H), 2.33-
2.11 (m, 2H), 1.95
(q, J=7.0Hz, 1H), 1.63-1.55 (m, 8H), 1.50-1.40 (m, 2H), 1.38-1.33 (m, 4H),
1.29 (d, J=7.0Hz,
3H), 0.83 (d, J=7.0Hz, 6H). LCMS [M-FH] = 844.3.
[0623] The following ATAC6 compound and ATAC7 compound in TABLE 22 can be
prepared
using a method similar to that described above for ATAC5.
TABLE 22
Compound Structure Name M+1
ATAC6 0 H 0 CH3 H H2N 2,5-dioxopyrrolidin-1 -y1 7-
872
r N, \ 4
(((S)-1-(((S)-1-((4-(((((4-
HO amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo [4,5-0 qu inolin-2-
yl)methyl)(ethyl)carb amoyl)o
xy)methyl)phenyl)amino)-1-
oxopropan-2-yl)amino)-3 -
methyl-l-oxobutan-2-
yl)amino)-7-oxoheptanoate
ATAC7 H2N ,HT0 2,5-dioxopyrrolidin-1 -y1 7-
958
0 H2N (((S)-1-(((S)-1-((4-(((((4-
r N, \ di amino-1-(2-hydroxy-2-
HC>(,.1...,N
methylpropy1)-1H-
i
imidazo [4,5-0 qu inolin-2-
yl)methyl)(ethyl)carb amoyl)o
xy)methyl)phenyl)amino)-1 -
271
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
oxo-5-ureidopentan-2-
yl)amino)-3-methyl-1-
oxobutan-2-yl)amino)-7-
oxoheptanoate
EXAMPLE 5
Synthesis of ATAC8, ATAC9, and ATAC10
[0624] This example shows synthesis of Perfluorophenyl 6-(((S)- 1-(((S)- 1-((4-
((((5-(2-amino-3-
pentylquino lin-5- yl)p entyl)c arbamo yl)o xy)methyl)phenyl)amino)- 1-o
xoprop an-2- yl)amino)-3 -
methyl- 1-o xobutan-2- yl)amino)-6-o xohexano ate (AT AC 8), perfluorophenyl 7-
(((S)- 1-(((S)- 1-
((4-(((((4-amino- 1-(2-hydro xy-2-methylprop y1)- 1H-imidazo [4,5-c] quino lin-
2-
yl)methyl)(ethyl)c arbamo yl)o xy)methyl)phenyl)amino)- 1-o xoprop an-2- yl)
amino)-3 -methyl- 1-
oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC9), and perfluorophenyl 7-(((S)- 1-
(((S)- 1-((4-
(((((4-amino- 1-(2-hydro xy-2-methylprop y1)- 1H-imidazo [4,5-c] quino lin-2-
yl)methyl)(ethyl)c arbamo yl)o xy)methyl)phenyl)amino)- 1-o xo-5-ureidopentan-
2- yl)amino)-3 -
methyl- 1-o xobutan-2- yl)amino)-7-o xoheptano ate (AT AC 10).
TFA
NH2
0 CH3 N
F O N AN )H.r N
= H
0 0 0 0./
0
ATAC8
Step A: Preparation of ATAC8
NCI F OH
NH2
0 CH3 N
HO ININ))NH
0 r1
FF
0 0 0,
DIC
0
TFA
NH2
ENi
0
0 0 el 0,,i
0
ATAC8
272
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
[0625] To a stirred solution of 6-(((S)-1-(((S)-1-((4-((((5-(2-amino-3-
pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-
1-oxobutan-
2-yl)amino)-6-oxohexanoic acid hydrochloride (168 mg, 0.215 mmol) and
pentafluorophenol
(158 mg, 0.86 mmol) in DMF (3 ml) was added N,N'-diisopropylcarbodiimide
(0.166 ml, 1.07
mmol) dropwise and the reaction mixture was stirred at room temperature for
6h. The reaction
mixture was concentrated and the residue was dissolved in DMSO and purified by
reverse phase
chromatography [water/acetonitrile (0.1% TFA)] from 10% followed by a gradient
from 20 to
80%. Pure fractions were combined to give perfluorophenyl 6-(((S)-1-(((S)-1-
((4-((((5-(2-amino-
3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate 2,2,2-trifluoroacetate (122 mg)
as a white solid.
1H NMR (DMSO-d6) 8 13.5 (s, 1H), 9.92 (s, 1H), 8.35 (bs, 3H), 8.17 (d,
J=7.0Hz, 1H), 7.87 (d,
J=7.0Hz, 1H), 7.64-7.52 (m, 4H), 7.32-7.18 (m, 4H), 4.91 (s, 2H), 4.37 (t,
J=7.0Hz, 1H), 4.19 (t,
J=7.0Hz, 1H), 3.60-3.50 (m, 4H), 2.97 (m, 4H), 2.79 (t, J=7.0Hz, 2H), 2.74 (t,
J=7.0Hz, 2H),
2.31-2.22 (m, 2H), 1.96 (q, J=7.0Hz, 1H), 1.71-1.51 (m, 8H), 1.45-1.38 (m,
2H), 1.40-1.27 (m,
9H), 0.90-0.80 (m, 9H). LCMS [M-FH] = 913.4.
[0626] The following compounds in TABLE 23 can be prepared using a method
similar to that
described in above for ATAC8.
TABLE 23
Compound Structure Name M+1
ATAC9¨N F perfluorophenyl 7-(((S)-1-(((S)-
941
0
F F _o 0 0 1410 0 FC.IN \
HC 1-((4-(((((4-amino-1-(2-
hydroxy-2-methylpropy1)-1H-
imidazo [4,5-0 quinolin-2-
yl)methyl)(ethyl)carbamoyl)oxy
)methyl)phenyl)amino)-1-
oxopropan-2-yl)amino)-3 -
methyl-l-oxobutan-2-yl)amino)-
7-oxoheptanoate
ATAC10 H2N.Hr0
perfluorophenyl 7-(((S)-1-(((S)- 1027
FI2N 1-((4-(((((4-amino-1-(2-
F 0 Erõ, Erõ,
¨N
F
_ 0 0 0 140 0 N hydroxy-2-methylpropy1)-1H-
F r
HO>r) imidazo [4,5-c] qu inolin-2-
yl)methyl)(ethyl)carb amoyl)oxy
)methyl)phenyl)amino)-1-oxo-5-
ureidopentan-2-yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-
273
CA 03049791 2019-07-09
WO 2018/140831
PCT/US2018/015607
7-oxoheptanoate
EXAMPLE 6
Synthesis of ATAC11
[0627] This example shows the synthesis of N-((4-amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo[4,5-c]quinolin-2-y1)methyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-
N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (ATAC11).
0 0 0
6C)N C)0C)ON 1,.::,N NH2
\ H
) N/ \ N
0 H3C //c........
OH lik
ATAC11
Step A. Preparation of ATAC11
0
c / EN l gardiquimod
DCC, DCM/ACN
0 0 0
0 0 0
.__.IC)LN-'''\.=-' ',...--''-o=-'\..--. ',..../'-o=-",---jeLN r-,N NH2
H
0 H3C
/c...,
OH
ATAC11
[0628] A solution of MAL-PEG4-acid (265.7 mg, 0.638 mmol) and N,N'-
dicyclohexylcarbodiimide (DCC, 144.8 mg, 0.702 mmol) in dry dichloromethane /
acetonitrile
(1:1,5 mL) was stirred at room temperature for lh, followed by addition of
compound 1 (100 mg,
0.319 mmol) in one portion. After 72h of stirring, volatile organics were
removed under vacuum.
The residue obtained was purified by flash column chromatography on silica
gel, eluting with
step gradients of methanol in dichloromethane at a ratio of v/v 1:20, 1:15,
and 1:9, to afford the
target product N-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-
c]quinolin-2-
yl)methyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-ethyl-
3,6,9,12-
tetraoxapentadecan-15-amide (80 mg, 35% yield) as white colored foamy solid
oil. 1H NMR
(300 MHz, CDC13) 8 8.40 ¨ 7.82 (br m, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.44 (t,
J=7.5 Hz, 1H), 7.30
(t, J=7.4 Hz, 1H), 6.76 ¨ 6.28 (br m, 2H), 4.82 ¨ 4.32 (br m, 2H), 4.08 ¨ 3.64
(br m, 6H), 3.54 (br
274
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
s, 14H), 3.31 (br s, 3H), 2.63 (br s, 2H), 2.38 (t, J=6.9 Hz, 2H), 1.27 (br s,
4H), 1.20 ¨ 0.68 (br m,
5H). MS (ESI+) m/z 712 (M+1), 734 (M+Na).
EXAMPLE 7
Synthesis of ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18,
ATAC19, ATAC20, and ATAC21
[0629] This example shows the synthesis of N-(5-(2-amino-3-pentylquinolin-5-
yl)penty1)-1-(3-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-
15-amide
(ATAC12), 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(3-
pentylquinolin-2-
y1)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC13), 1-(3-(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-
yl)propanamido)-N-(1-isobuty1-1H-imidazo[4,5-c]quinolin-4-y1)-3,6,9,12-
tetraoxapentadecan-
15-amide (ATAC14), 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
methyl-N-(2-
(3-(7-methylbenzo[1,2-d:3,4-0bis(thiazole)-2-yl)ureido)ethyl)-3,6,9,12-
tetraoxapentadecan-15-
amide (ATAC15), (5)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
(1-((7-
methylbenzo[1,2-d:3,4-0bis(thiazole)-2-yOamino)-1-oxo-3-phenylpropan-2-y1)-
3,6,9,12-
tetraoxapentadecan-15-amide (ATAC16), N-(benzo[d]thiazol-2-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-N-((8-hydroxyquinolin-7-y1)(4-
(trifluoromethoxy)phenyOmethyl)-
3,6,9,12-tetraoxapentadecan- 15-amide (ATAC17), N-
((2R,3R,3a5,7aR,9R,10R,10aS,14aR)-2,9-
bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-5,10,12-trihydroxy-5,12-
dioxidodecahydrodifuro[3,2-
d:3',2'-j][1,3,7,9,2,8]tetra-oxadiphosphacyclododecin-3-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC18), N-
((2R,3R,3 aS,7 aR,9R,10R,10aS ,14aR)-2,9-bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-
10-hydroxy-
5,12-dimercapto-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-
A [1,3,7,9,2,8]tetraoxadiphosphacyclododecin-3-y1)-1-(3-(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC19), N-(9-
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo- 1H-purin-9(6H)-y1)-3
,5,10,12-
tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'A [1,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC20), and N-(9-
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo- 1H-purin-9(6H)-y1)-3
,5,10,12-
tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-
A [1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-
dioxo-2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC21).
275
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
H3C
NH2
___NµIN C)0C)ON
\ H H
0
ATAC12
Step A. Preparation of ATAC12
H3C
NH2 H2N Mal-PEG4-NHS
1 _______________________________________ ..-
N
DCM
H3C
NH2
H
0
ATAC12
[0630] To a stirred solution containing 100 mg (0.33 mmol) of 5-(5-
aminopenty1)-3-
pentylquinolin-2-amine in 13 mL of CH2C12 under N2 was added a solution of MAL-
PEG4-NHS
[CAS No 756525-99-2] (171 mg, 0.33 mmol) in 3 mL of CH2C12 by syringe pump
over 90 mins.
The reaction mixture was stirred at room temperature for 16h then evaporated
to afford a residue
which was purified by silica gel chromatography (CombiFlash Gold (12g):
CH2C12/CH3OH/NH4OH) to afford a light yellow syrup which was dissolved in 5 mL
of CH3CN
and lyophilized to provide 164 mg of the desired compound. 1H NMR (CD30D) 8
7.95 (s, 1H),
7.38 (s, 1H), 7.37 (s, 1H), 7.07 (t, J=8.5Hz, 1H), 6.78 (s, 2H), 3.75 (t,
J=6.0Hz, 2H), 3.65 (t,
J=6.0Hz, 2H), 3.59-3.52 (m, 12H), 3.46 (t, J=5.5Hz, 2H), 3.28 (t, J=7.5Hz,
2H), 3.18 (t, J=7.5Hz,
2H), 2.98 (t, J=8.5Hz, 2H), 2.67 (t, J=7.5Hz, 2H), 2.44 (t, J=7.0 Hz, 2H),
2.40 (t, J=7.0 Hz, 2H),
1.76-1.68 (m, 4H), 1.58-1.52 (m, 2H), 1.46-1.40 (m, 6H), 0.94 (t, J=7.0Hz,
3H). (MS (ESI+) m/z
698 (M+1).
[0631] The following compounds in TABLE 24 can be prepared using a method
similar to that
as described above for ATAC12.
TABLE 24
276
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
Compound Structure Name M+1
ATAC13
N-111 1-(3-(2,5-dioxo-2,5-dihydro-1H-
613
0 0
I
pyrrol-1-yl)propanamido)-N-(3-
0
HaC
pentylquinolin-2-y1)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC14 1-(3-(2,5-dioxo-2,5-dihydro-1H- 639
0
H Hn-- pyrrol-1-yl)propanamido)-N-(1-
0
isobuty1-1H-imidazo[4,5-
c]quinolin-4-y1)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC15 JOO 1-(3-(2,5-dioxo-2,5-dihydro-1H-
720
H ! r!!
0 pyrrol-1-yl)propanamido)-N-
methyl-N-(2-(3-(7-
methylbenzo[1,2-d:3,4-
dalbis(thiazole)-2-yOureido)ethyl)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC16 (S)-1-(3-(2,5-dioxo-2,5-dihydro-
635
o 1H-pyrrol-1-y 1)propanamido)-N-
,tz. 0 o hi
N
0
(1-((7-methylbenzo[1,2-d:3,4-
sy:
dlbis(thiazole)-2-yl)amino)-1-
oxo-3-phenylpropan-2-y1)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC17 N-(benzokflthiazol-2-y1)-1-(3-
734
o I
S (2,5-dioxo-2,5-dihydro-1H-pyrrol-
o
IN1 - C)Hr 1-yl)propanamido)-N-((8-
0 OH
0 hydroxyquinolin-7-y1)(4-
ocF3 (trifluoromethoxy)phenyl)methyl)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC18 N- 1088
HC>9-hc49 k(1 XNH
HO o : 0 ((2R,3R,3a5,7aR,9R,10R,10aS,14
HN
2H,T-Njk aR)-2,9-bis(2-amino-6-oxo-1H-
0
purin-9(6H)-y1)-5,10,12-
trihydroxy-5,12-
dioxidodecahydrodifuro[3,2-
277
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
d:3',2'-j][1,3,7,9,2,8[tetra-
oxadiphosphacyclododecin-3-y1)-
1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC19 <;,(c)NH N-
1120
Hcj_./P-01c4 ((2R,3R,3aS,7aR,9R,10R,10aS,14
roA0,--- ON
N'NokTX, NSF' aR)-2,9-bis(2-amino-6-oxo-1H-
purin-9(6H)-y1)-10-hydroxy-5,12-
dimercapto-5,12-
dioxidodecahydrodifuro113,2-
d:3',2'-
j]111,3,7,9,2,8[tetraoxadiphosphacy
clododecin-3-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC20 N-(9-
1073
p 0(51 H 0
IcL N ((2R,3R,3aS,7aR,9R,10R,10aS,14
H'N aR)-9-(2-amino-6-oxo-1H-purin-
trN,>
9(6H)-y1)-3,5,10,12-tetrahydroxy-
5,12-dioxidodecahydrodifuro113,2-
d:3',2'-j]111,3,7,9,2,8[tetra-
oxadiphosphacyclododecin-2-y1)-
9H-purin-6-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC21 N-(9-
1073
P kin41 H 0
F01>'- N ((2R,3R,3aS,7aR,9R,10R,10aS,14
OH aR)-9-(2-amino-6-oxo-1H-purin-
0 9(6H)-y1)-3,5,10,12-tetrahydroxy-
5,12-dioxidodecahydrodifuro113,2-
d:3',2'-
j][1,3,7,9,2,8[tetraoxadiphosphacy
clododecin-2-y1)-9H-purin-6-y1)-
278
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-
3,6,9,12-tetraoxapentadecan-15-
amide
EXAMPLE 8
Synthesis of ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28,
ATAC29, ATAC30, and ATAC31
[0632] This example shows the synthesis of 4-((S )-2-((S)-2-(6-(2,5-dioxo-2,5-
dihydro-1H-
p yrrol- 1-yl)hexanamido)-3 -methylbutanamido)prop anamido)benzyl ((4-amino- 1-
(2-hydro xy-2-
methyl-prop y1)- 1H-imidazo[4,5-c] quinolin-2-yl)methyl)(ethyl)c arbamate
(ATAC22), 4-((S)-2-
((S )-2-(6-(2,5-dio xo-2,5-dihydro- 1H-pyrrol- 1-yl)hexanamido)-3 -methyl-
butanamido)propanamido)benzyl (5-(2-amino-3-pentylquinolin-5-yl)penty1)-
carbamate
(ATAC23), 4-((S )-2-((S )-2-(6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol- 1-
yl)hexanamido)-3-
methylbutan-amido)-5-ureidopentanamido)benzyl- (5-(2-amino-3 -pentylquino lin-
5-yl)penty1)-
c arbamate (ATAC24), 4-((S )-2-((S )-2-(6-(2,5-dio xo-2,5-dihydro- 1H-pyrrol-
1-yl)hexanamido)-3 -
methylbutanamido)-5-ureidopentanamido)benzyl((4-amino- 1-(2-hydro xy-2-
methylprop y1)- 1H-
imidazo[4,5-c] quinolin-2-yl)methyl)(ethyl)c arbamate TFA salt (AT AC25), 2-(3
-12-11\1-
Methyl(1p- RS )-2-1(S )-2-[6-(2,5-dio xo- 1H-pyrrol- 1-yOhexano ylamino] -3 -
methylbutyrylamino } -
5-ureidovalerylamino]phenyl} metho xyc arbonyl) amino] ethyl } ureido)-7-
methyl- 1,6-dithia-3 ,8-
diaza-as-indacene (ATAC26), 2-1 R8-Hydroxy-7-quinoly1)(p-
trifluorometho xyphenyOmethyl] (1p- RS )-2-1(S)-2-[6-(2,5-dio xo- 1H-pyrrol- 1-
yOhexano ylamino] -
3 -methylbutyrylamino } -5-ureidovalerylamino]phenyl} metho xyc arbonyl) amino
} - 1,3 -
benzothiazole (ATAC27), (1R,6R,8R,95 ,10S,15R,17R,18S )- 1841 p- RS )-2-1 (S )-
2-[6-(2,5-Dioxo-
1H-p yrrol- 1-yl)hexano ylamino] -3 -methylbutyrylamino } -5-
ureido valerylamino]phenyl } metho xyc arbonylamino)- 8,17-bis(2-amino-6-o xo-
1,9-dihydropurin-
9-y1)-3,12-dihydro xy-9-hydro xy-2.4.7.11.13.16-hexao xa-3k5 .1215-
dipho sphatricyclo[13.3Ø06,10]octadecane-3,12-dione (ATAC28),
(1R,6R,8R,95,10S ,15R,17R,18S )4841 p-RS)-2-1 (S)-2-[6-(2,5-Dioxo-1H-pyrrol-1-
yl)hexano ylamino] -3 -methylbutyrylamino
}propionylamino]phenyl}methoxycarbonylamino)-
8,17-bis(2-amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-9-hydroxy-
2.4.7.11.13.16-
hexaoxa-3k5.1215-diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione
(ATAC29),
(1R,6R,8R,95,10S ,15R,17R,18S )4841 p-RS)-2-1 (S)-2-[6-(2,5-Dioxo-1H-pyrrol-1-
yl)hexano ylamino] -3 -methylbutyrylamino } -5-
ureido valerylamino]phenyl } metho xyc arbonylamino)- 8,17-bis(2-amino-6-o xo-
1,9-dihydropurin-
279
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
9- y1)-9-hydro xy-3,12-dimerc apto-2.4.7 .11.13 .16-hexao xa-3 X5 .12X5 -
dipho sphatricyclo [13 .3 Ø06,10]octadecane-3,12-dione (ATAC30), and 1p- )-2-
{ (S )-246-(2,5-
Dio xo- 1H-pyrrol- 1- yflhexano ylamino} -3 -methylb utyrylamino } -5-
ureidovalerylaminolphenyl } methyl 9-1 (1S ,6R,8R,9S ,10S ,15R,17R,18S )-8-(2-
amino-6-oxo- 1,9-
dihydropurin-9- y1)-3,12-dihydro xy-9,18-dihydro xy-3,12-dio xo -2.4.7 .11.13
.16-hexao xa-
3 X5 .12X5-diphosphatricyclo [13 .2.1.06,10]o ctadec-17- yl} -9a- adenineec
arbo xylate (ATAC31).
0
0 1. NH2
NN N N
H3C
0 0 CH3
OH
ATAC22
Step A: Preparation of ATAC22
NO
o A Compound 1
0 0 O
cr1LN rL).N DIPEA, DMF, r t
0 0 CH3
MC-Val-Ala-PAB-PNP
cri0 r\rriN= oNr.N NH2
0 0
N \ N
H3C
0 0 CH3
OH
ATAC22
[0633] A solution of compound 1(150 mg, 0.479 mmol) and N,N'-
diisopropylethylamine (145.4
mg, 1.437 mmol) in dry DMF was stirred at room temperature for 5 min.,
followed by addition of
maleimidocaproyl-valine-alanine-p-aminobenzyl alcohol p-nitrophenyl-carbonate
(MC-Val-Ala-
PAB-PNP, 343.6 mg, 0.527 mmol). After stirring for 24 h, volatile organics
were removed under
vacuum. The residue obtained was triturated with dry acetonitrile. The
precipitated solid was
collected by filtration, washed with acetonitrile and dried under vacuum to
obtain unreacted MC-
Val-Ala-PAB-PNP (130 mg) as beige solid. The filtrate and washings were
combined and
concentrated under vacuum. The residue obtained was purified by flash column
chromatography
on silica gel, eluting with step gradients of Me0H in dichloromethane at a
ratio of v/v 1:20, 1:15,
280
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
and 1:10, to afford the target product mc-Val-Ala-PAB-GDQ (70 mg, 18% yield)
as beige
colored foamy solid. 1H NMR (DMSO-d6) 8 10.1 ¨ 9.75 (br m, 1H), 8.58 ¨ 8.24
(br m, 1H), 8.15
(d, J=6.6 Hz, 1H), 8.01 (br s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.4 Hz,
1H), 7.65 ¨ 7.48 (m,
2H), 7.46 ¨7.34 (m, 2H), 7.29 (br s, 1H), 7.18 (br s, 1H), 6.99 (s, 2H), 5.03
(br s, 2H), 4.96 (br s,
1H), 4.72 (br s, 1H), 4.48 ¨ 4.26 (m, 1H), 4.26 ¨ 4.04 (m, 1H), 2.22 ¨ 2.02
(m, 2H), 2.02 ¨ 1.80
(m, 1H), 1.58 ¨ 1.37 (m, 4H), 1.36 ¨0.92 (br m, 15H), 0.92 ¨0.53 ( br m, 7H).
MS (ESI+) m/z
826 (M+1).
[0634] The following ATAC30, ATAC31, ATAC32, ATAC33, ATAC34, ATAC35, ATAC36,
ATAC37, ATAC38, ATAC39, ATAC40, ATAC41, and ATAC42 are prepared using a method
similar to that described above for ATAC29.
ATAC23: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-
methyl-
butanamido)propanamido)benzyl (5-(2-amino-3-pentylquinolin-5-yl)penty1)-
carbamate
0
cro H r\cr EN1,)N1
0 0 1
0 0 u1-13H 0 1 N
H3C
NH2
ATAC23
[0635] 1H NMR (CD30D) 8 8.35 (s, 1H), 7.63 (t, J=8.5Hz, 1H), 7.55 (d, J=8.0Hz,
1H), 7.47 (d,
J=8.0Hz, 1H), 7.33 (d, J=8.0Hz, 1H), 7.27(d, J=8.0Hz, 1H), 6.78 (s, 2H), 5.00
(s, 2H), 4.46 (q,
J=7.0Hz, 2H), 4.13 (d, J=7.0Hz, 1H), 3.47-3.4 (m, 3H), 3.17 (t, J=7.0Hz, 2H),
3.05 (t, J=7.0Hz,
2H), 2.75 (t, J=7.5Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 2.07 (q, J=7.0Hz, 1H), 1.72-
1.51 (m, 10H),
1.46-1.35 (m, 8H), 1.32-1.26 (m, 3H), 1.00-0.92 (m, 9H). LCMS [M+H] = 812.4.
ATAC24: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)hexanamido)-3-
methylbutan-amido)-5-ureidopentanamido)benzyl-(5-(2-amino-3-pentylquinolin-5-
yl)penty1)-
carbamate
281
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
0
0
o oAH c---f 0
' NõA)crkl,AN
H H
0 0
H3C N
NH2
yH
H2NO
ATAC24
1H NMR (DMSO-d6) 8 13.5 (bs, 1H), 10.0 (s, 1H), 8.40 (m, 3H), 8.07 (d,
J=7.5Hz, 1H), 7.80 (d,
J=8.5 Hz, 1H), 7.6-7.5 (m, 4H), 7.35-7.25 (m, 2H), 6.01 (m, 1H), 5.42 (s, 1H),
4.89 (s, 2H), 4.41
(q, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 3.10 ¨ 2.90 (m, 6H), 2.75 (t,
J=7.5Hz, 2H), 2.27 (t,
J=7.5Hz, 2H), 2.07 (q, J=7.0Hz, 1H), 1.72-1.51 (m, 10H), 1.46-1.35 (m, 8H),
1.32-1.26 (m, 3H),
1.00-0.92 (m, 9H). LCMS [M+H] = 898.
ATAC25: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-
methylbutanamido)-5-ureidopentanamido)benzyl((4-amino-1-(2-hydroxy-2-
methylpropy1)- 1H-
imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate TFA salt
0
0 NH2
cf 0 0 0LX)L )\11-=-N
N N N \ N
H H
0 0
OH
NH
H2NO TFA
ATAC25
1H NMR (DMSO-d6) 8 13.4 (bs, 1H), 9.99 ¨ 9.89 (br m, 1H), 9.09 ¨ 8.40 (m, 3H),
8.07 (d,
J=7.5Hz, 1H), 7.80 (d, J=8.5 Hz, 1H), 7.68 (t, J=8.0 Hz, 1H), 7.59 (bs, 1H),
7.51 (t, J=8.5Hz,
1H), 7.46 ¨ 7.14 (m, 2H), 7.00 (s, 1H), 5.99 (br s, 1H), 5.05 (br s, 1H), 4.95
(br s, 1H), 4.37 (q,
J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 3.37 (t, J=7.0Hz, 2H), 3.03 ¨ 2.93 (m,
2H), 2.22 ¨ 2.07 (m,
2H), 1.99 ¨ 1.92 (m, 1H), 1.75 ¨ 1.05 (br m, 20H), 0.85 (d, J=8.5Hz, 3H), 0.81
(d, J=8.5Hz, 3H).
MS (ESI+) m/z 912.5 (M+1).
TABLE 25
Compound Structure Name
M+1
282
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
ATAC26 0 H H ry 2-
(3-{24N-Methyl({ p-(S)-2-{ (S)-246-(2,5- 921
cri()crri.(N
0 H 0 H dioxo-1H-pyrrol-1-yl)hexanoylamino]-3-
\H
methylbutyrylamino1-5-
ureidovalerylamino]phenyl methoxycarbonyl
)amino]ethyllureido)-7-methy1-1,6-dithia-3,8-
diaza-as-indacene
ATAC27 OCF, 2- { R8-Hydroxy-7-quinoly1)(p-
1067
0 01-1 trifluoromethoxyphenyl)methyl]({p-
RS)-2-
r_f
N; (S)-246-(2,5-dioxo-1H-pyrrol-1-
0 H 0
\im 0 yl)hexanoylamino]-3-methylbutyrylamino
I -5-
NH
ureidovalerylamino]phenyl I methoxycarbonyl
)amino -1,3-benzothiazole
ATAC28 1r H2N 0
(1R,6R,8R,9S,10S,15R,17R,18S)-18-({p-(S)- 1289
4,1-11:r
H0,9 aL N N NH2 HN
HO - 2- { (S)-2-[6-(2,5-Dioxo-1H-pyrrol-1
H
P lc,17
H 0
RA HN,_0,o-NrANY----,4 yl)hexanoylamino]-3-methylbutyrylamino1-5-
2NzNr,,,r) -6 0H r8- 0 0
O
ureidovalerylamino]phenyl I methoxycarbonyl
amino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-9-y1)-3,12-dihydroxy-9-
hydroxy-2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-
3,12-dione
ATAC29 0
(1R,6R,8R,9S,10S,15R,17R,18S)-18-({p4(S)- 1203
HO Foirclov4DN N NH 2
2- { (S)-246-(2,5-Dioxo-1H-pyrrol-1-
p..,0 NN 0 õ..õ. Edlor:,CHy 0 N 0
yl)hexanoylamino]-3-
H21,1z,NrN,1 C> 6,0H ej
0
1r,'LN methylbutyrylamino Ipropionylamino]phenyl
methoxycarbonylamino)-8,17-bis(2-amino-6-
oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-9-
hydroxy-2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-
3,12-dione
ATAC30 0 (1R,6R,8R,95,105,15R,17R,185)-18-
({p4(S)- 1321
N NH241
HO HS
2- { (S)-246-(2,5-Dioxo-1H-pyrrol-1-
HN
0
H 19 0 N
ICC?(0_pr =* 0 y 1)hexanoylamino]-3-methylbutyrylamino1-5-
H2NH,Nr,Nir crai
0
ureidovalerylamino]phenyl I methoxycarbonyl
amino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-9-y1)-9-hydroxy-3,12-
dimercapto-2.4.7.11.13.16-hexaoxa-35.1215-
283
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
diphosphatricyclol13.3Ø06,10loctadecane-
3,12-dione
ATAC31 HN:10 *Q H`.( 1134(S)-2-{(S)-246-(2,5-Dioxo-1H-pyrrol-1-
1274
5 yl)hexanoylaminol-3-methylbutyrylaminol-5-
I
HO H-9 1
0 4sN11.)"
EI;1 0 0 CC71 ureidovalerylaminolphenyllmethyl 9-
H2NErytNN 07;0H
{(1S,6R,8R,9S,10S,15R,17R,18S)-8-(2-
amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-
dihydroxy-9,18-dihydroxy-3,12-dioxo-
2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclol13.2.1.06,10loctadec-17-
y11-9a-adenineecarboxylate
EXAMPLE 9
Synthesis of ATAC32
[0636] This example shows the synthesis of 1-16-1(17-Amino-3-(2-hydroxy-2-
methylpropy1)-
3.5.8-triazatricyclo[7.4Ø02,6}trideca-1(9),2(6),4,7,10,12-hexaen-4-y1}
methyl)-N-ethylamino} -6-
oxohexy1}-1H-pyrrole-2,5-dione (ATAC32).
0
0
0 ) N H3C N
OH *
ATAC32
Step A: Preparation of ATAC32
0
JN(
0 0
0
NLOH \NNH2
0 H3C
OH
ATAC32
[0637] To an ice-cold solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)hexanoic acid (0.034
g, 0.16 mmol) in DCM (0.800 ml) was added 1-chloro-N,N,2-trimethylprop-1-en-1-
amine (0.021
mL, 0.160 mmol) dropwise. This was stirred at 0 C for lh then added to an ice-
cold mixture of
compound 1(50 mg, 0.160 mmol) and triethylamine (66.7 i.tt, 0.479 mmol) in DCM
(800 lL).
Overall molarity 0.1 M. The mixture was stirred to room temperature overnight
and then
284
CA 03049791 2019-07-09
WO 2018/140831 PCT/US2018/015607
chromatographed (DCM to 20% Me0H/DCM) without work-up. Fractions containing
product
were pooled and evaporated then dissolved in 1 mL of acetonitrile and treated
with 0.1 mL of
trifluoroacetic acid. The resulting material was evaporated to an oil then
redissolved in CH3CN
and lyophilized the sample to give ATAC32 (65 mg) as a white solid. 1H NMR
(400 MHz,
(DMSO-d6) 8 13.3 (s, 1H), 8.54 ¨ 8.50 (m, 3H), 7.81 (d, J=8.5 Hz, 1H), 7.76
(d, J=7.5 Hz, 1H),
7.51 (d, J=7.5 Hz, 1H), 6.99 (s, 1H), 6.95 (s, 1H), 3.51 (q, J=7.0 Hz, 2H),
3.43-3.31 (m, 3H),
2.36-2.30 (m, 2H), 1.54-1.41 (m, 4H), 1.25-1.00 (m, 10H). 19F NMR (DMSO-d6) 8 -
74Ø LCMS
[A4 + H]' = 507.1.
EXAMPLE 10
Synthesis of ATAC33
[0638] This example shows the synthesis of 1-1 [4-(164(17-Amino-3-(2-hydroxy-2-
methylpropy1)-3.5.8-triazatricyclo .4Ø02,6}trideca-1(9),2(6),4,7,10,12-
hexaen-4-y1} methyl)-N-
ethylamino}-6-oxohexylamino }carbonyl)cyclohexyl} methyl} -1H-pyrrole-2,5-
dione (ATAC33).
0
t.I.\µ1rH0
0 NH2
0 ) N \ N
H3C
OH 41/
ATAC33
Step A: Preparation of ATAC33
0
0
0 0
0
compound 1
0
0
NH2
0
0 ) N \ N
H3C
OH
ATAC33
[0639] To a stirred solution of 1-(4-amino-2-((ethylamino)methyl)-1H-
imidazo[4,5-c]quinolin-1-
y1)-2-methylpropan-2-ol (100 mg, 0.319 mmol) in DCM (10 mL) under nitrogen was
added via a
syringe pump a solution of 2,5-dioxopyrrolidin-1-y16-(4-((2,5-dioxo-2,5-
dihydro-1H-pyrrol-1-
285
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 285
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 285
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
